Antibodies specifically binding PD-1, TIM-3 or PD-1 and TIM-3 and their uses

ABSTRACT

The present invention relates to antibodies specifically binding PD-1, TIM-3 or PD-1 and TIM-3, polynucleotides encoding the antibodies or fragments, and methods of making and using the foregoing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/250,095 filed 3 Nov. 2015, the entire contents of theaforementioned applications are incorporated herein by reference intheir entireties.

SEQUENCE LISTING

This application contains a Sequence Listing submitted via EFS-Web, theentire content incorporated herein by reference in its entirety. TheASCII text file, created on 28 Oct. 2016, is named JBI5071USNP_ST25.txtand is 416 kilobytes in size.

FIELD OF THE INVENTION

The present invention relates antibodies specifically binding PD-1,TIM-3 or PD-1 and TIM-3, polynucleotides encoding the antibodies orfragments, and methods of making and using the foregoing.

BACKGROUND OF THE INVENTION

The immune system is tightly controlled by a network of costimulatoryand co-inhibitory ligands and receptors. These molecules providesecondary signals for T cell activation and provide a balanced networkof positive and negative signals to maximize immune responses againstinfection and tumors, while limiting immunity to self (Wang et al.,(Epub Mar. 7, 2011) J Exp Med 208(3):577-92; Lepenies et al., (2008)Endocr Metab Immune Disord Drug Targets 8:279-288).

Immune checkpoint therapy, targeting co-inhibitory pathways in T cellsto promote antitumor immune responses, has led to advances in clinicalcare of cancer patients.

PD-1 is a negative immune checkpoint molecule that suppresses CD4⁺ andCD8⁺ T cell functions in the tumor microenvironment (TME). PD-1engagement with its ligands (PD-L1 and PD-L2) drives T cell anergy andexhaustion in tumors by inhibiting multiple pathways downstream of the Tcell receptor signaling, resulting in decreased T cell survival, growthand proliferation, compromised effector function, and alteredmetabolism. Preclinical studies have demonstrated that the PD-1 pathwayblockade can reverse T cell exhaustion and stimulate anti-tumorimmunity.

The PD-1 pathway hence contributes to downregulation of T cell functionsin the (TME) and evasion of tumors via immune destruction. In the TME,exhausted T cells, in addition to expressing high levels of PD-1,express other inhibitory receptors including CTLA-4, TIM-3, LAG-3,CD244, TIGIT and CD160 (see e.g., Pauken & Wherry; 2015, Trends inImmunology 36(4): 265-276).

TIM-3 is a transmembrane receptor that is expressed on Th1 (T helper 1)CD4⁺ cells and cytotoxic CD8⁺ T cells that secrete IFN-γ. TIM-3 isgenerally not expressed on naïve T cells but rather upregulated onactivated, effector T cells. TIM-3 has a role in regulating immunity andtolerance in vivo (see Hastings et al., (2009) Eur J Immunol39(9):2492-501).

PD-1 antibodies have been described for example in: U.S. Pat. Nos.5,897,862 and 7,488,802, and in Int. Patent Publ. Nos. WO2004/004771,WO2004/056875, WO2006/121168, WO2008/156712, WO2010/029435,WO2010/036959, WO2011/110604, WO2012/145493, WO2014/194302,WO2014/206107, WO2015/036394, WO2015/035606, WO2015/085847,WO2015/112900 and WO2015/112805.

TIM-3 antibodies have been described for example in: Monney et al.,Nature (2002) 415(6871):536-41, and in Int. Patent Publ. Nos.WO2011/155607, WO2013/006490 and WO2015/117002.

Combinations with TIM-3 antibody and a PD-L1 antibody have beenevaluated in for example in Int. Patent Publ. No. WO2011/159877.

While anti-PD-1/PD-L1 antibodies are demonstrating encouraging clinicalresponses in patients with multiple solid tumors, the response rates arestill fairly low, about 15%-20% in pretreated patients (Swaika et al.,(2015) Mol Immunol. doi: 10.1016/j.molimm.2015.02.009).

Therefore, there is a need for new therapeutics that inhibit theimmunosuppressive activity of checkpoint inhibitors such as PD-1 andTIM-3, to be used for cancer immunotherapy and treatment of otherconditions that would benefit from enhancement of an immune response,such as chronic infections.

BRIEF SUMMARY OF THE INVENTION

The invention provides an isolated antagonistic antibody specificallybinding PD-1, comprising a heavy chain complementarity determiningregion 1 (HCDR1), a HCDR2 and a HCDR3 of SEQ ID NOs: 82, 83 and 84,respectively, or SEQ ID NOs: 82, 83 and 85, respectively.

The invention also provides an isolated antagonistic antibodyspecifically binding PD-1, comprising a heavy chain complementaritydetermining region 1 (HCDR1), a HCDR2 and a HCDR3 of SEQ ID NOs: 82, 83and 84, respectively, and a light chain complementarity determiningregion 1 (LCDR1), a LCDR2 and a LCDR3 of SEQ ID NOs: 86, 87 and 88,respectively.

The invention also provides an isolated antagonistic antibodyspecifically binding PD-1, comprising a heavy chain complementaritydetermining region 1 (HCDR1), a HCDR2 and a HCDR3 of SEQ ID NOs: 82, 83and 85, respectively, and a light chain complementarity determiningregion 1 (LCDR1), a LCDR2 and a LCDR3 of SEQ ID NOs: 86, 87 and 88,respectively.

The invention also provides an isolated antagonistic antibodyspecifically binding PD-1, comprising certain HCDR1, HCDR2, HCDR3,LCDR1, LCDR2 and LCDR3 amino acid sequences as described herein.

The invention also provides an isolated antagonistic antibodyspecifically binding PD-1, comprising certain VH and VL amino acidsequences as described herein.

The invention also provides an isolated antagonistic antibodyspecifically binding TIM-3, comprising a heavy chain complementaritydetermining region 1 (HCDR1), a HCDR2 and a HCDR3 of SEQ ID NOs: 164,165 and 166, respectively.

The invention also provides an isolated antagonistic antibodyspecifically binding TIM-3, comprising the HCDR1, the HCDR2 and theHCDR3 of SEQ ID NOs: 164, 165 and 166, respectively, and the LCDR1, theLCDR2 and the LCDR3 of SEQ ID NOs: 167, 168 and 169 respectively.

The invention also provides an isolated antagonistic antibodyspecifically binding TIM-3, comprising certain HCDR1, HCDR2, HCDR3,LCDR1, LCDR2 and LCDR3 amino acid sequences as described herein.

The invention also provides an isolated antagonistic antibodyspecifically binding TIM-3, comprising certain VH and VL amino acidsequences as described herein.

The invention also provides an isolated antagonistic bispecificPD1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3.

The invention also provides an isolated antagonistic bispecificPD1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, comprising certainHCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, VH, VL, heavy chain or lightchain amino acids sequences as described herein.

The invention also provides an isolated antagonistic bispecificPD1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, wherein the first domaincomprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and theLCDR3 of SEQ ID NOs: 10, 14, 17, 23, 26 and 32, respectively, and thesecond domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 of SEQ ID NOs: 97, 105, 115, 124, 133 and 143,respectively.

The invention also provides an isolated antagonistic bispecificPD1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, wherein the first domaincomprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and theLCDR3 of SEQ ID NOs: 10, 14, 17, 23, 26 and 32, respectively, and thesecond domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 of SEQ ID NOs: 91, 99, 108, 118, 127 and 136,respectively.

The invention also provides an isolated antagonistic bispecificPD1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, wherein the first domaincomprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and theLCDR3 of SEQ ID NOs: 66, 67, 68, 69, 70 and 71, respectively, and thesecond domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 of SEQ ID NOs: 97, 105, 115, 124, 133 and 143,respectively.

The invention also provides an isolated antagonistic bispecificPD1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, wherein the first domaincomprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and theLCDR3 of SEQ ID NOs: 66, 67, 68, 69, 70 and 71, respectively, and thesecond domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 of SEQ ID NOs: 91, 99, 108, 118, 127 and 136,respectively.

The invention also provides an isolated antagonistic bispecificPD1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, wherein the first domaincomprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and theLCDR3 of SEQ ID NOs: 10, 14, 16, 723, 26 and 32, respectively, and thesecond domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 of SEQ ID NOs: 97, 105, 115, 124, 133 and 143,respectively.

The invention also provides an isolated antagonistic bispecificPD1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, wherein the first domaincomprises a heavy chain variable region (VH) of SEQ ID NO: 48 and alight chain variable region (VL) of SEQ ID NO: 56, and the second domaincomprises the VH of SEQ ID NO: 153 and the VL of SEQ ID NO: 162.

The invention also provides an isolated antagonistic bispecificPD1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, wherein the first domaincomprises the VH of SEQ ID NO: 48 and the VL of SEQ ID NO: 56, and thesecond domain comprises the VH of SEQ ID NO: 146 and the VL of SEQ IDNO: 156.

The invention also provides an isolated antagonistic bispecificPD1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, wherein the first domaincomprises the VH of SEQ ID NO: 64 and the VL of SEQ ID NO: 65, and thesecond domain comprises the VH of SEQ ID NO: 153 and the VL of SEQ IDNO: 162.

The invention also provides an isolated antagonistic bispecificPD1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, wherein the first domaincomprises the VH of SEQ ID NO: 64 and the VL of SEQ ID NO: 65, and thesecond domain comprises the VH of SEQ ID NO: 146 and the VL of SEQ IDNO: 156.

The invention also provides an isolated antagonistic bispecificPD1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, wherein the first domaincomprises the VH of SEQ ID NO: 48 and the VL of SEQ ID NO: 56, and thesecond domain comprises the VH of SEQ ID NO: 172 and the VL of SEQ IDNO: 173.

The invention also provides an immunoconjugate comprising the antibodyor antigen-binding portion thereof of the invention linked to atherapeutic agent or to an imaging agent.

The invention also provides a pharmaceutical composition comprising theantibody of the invention and a pharmaceutically accepted carrier.

The invention also provides a polynucleotide encoding the antibody VH,the antibody VL or the antibody VH and the antibody VL of the invention.

The invention also provides a vector comprising the polynucleotideencoding the antibody VH, the antibody VL or the antibody VH and the VLof the invention.

The invention also provides a host cell comprising the vector of theinvention.

The invention also provides a method of producing the antibody of theinvention, comprising culturing the host cell of the invention inconditions that the antibody is expressed, and recovering the antibodyproduced by the host cell.

The invention also provides a method of treating a cancer in a subject,comprising administering a therapeutically effective amount of theisolated antibody of the invention to the subject in need thereof for atime sufficient to treat the cancer.

The invention also provides a method of enhancing an immune response ina subject, comprising administering a therapeutically effective amountof the isolated antibody of the invention to the subject in need thereoffor a time sufficient to enhance the immune response.

The invention also provides an anti-idiotypic antibody binding to theantibody of the invention.

The invention also provides a kit comprising the antibody of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows that TIM-3 surface expression is elevated in tumors aftertreatment with anti-PD-1 antibodies. Balb/c mice with established CT26colon carcinoma tumors were treated biweekly with anti-PD-1 antibody orvehicle. Tumors were harvested at day 22 and TIM-3 expression wasevaluated on tumor-infiltrating T cells using flow cytometry. MFI: meanfluorescent intensity. PBS: control

FIG. 1B shows that TIM-3 surface expression is elevated on tumorinfiltrated lymphocytes (TIL) after treatment with anti-PD-1 antibodies.Balb/c mice with established MC38 colon carcinoma tumors were treatedbiweekly with anti-PD-1 antibody or vehicle. Geometric mean fluorescentintensity (gMFI) of TIM-3 expression on total CD8 TIL population isshown in vehicle treated (PBS) or anti-PD-1 antibody treated (PD-1)animals p=0.003 vehicle vs anti-PD-1 antibody treated groups.

FIG. 1C shows the relative frequency of TIM-3⁺ CD8 cells of total CD8⁺TILs in MC38 tumors harvested from mice treated with vehicle (PBS) oranti-PD-1 antibody (PD-1). p=0.045 vehicle vs anti-PD-1 antibody treatedgroups.

FIG. 2A shows that CD137 surface expression (gMFI) is elevated on TILsin MC38 colon carcinoma tumors in animals treated with anti-PD-1antibodies (PD-1 group) when compared to vehicle treated (PBS) group.p=0.005 vehicle vs anti-PD-1 antibody treated groups. Each pointrepresents one mouse. Data are representative of at least 2 independentexperiments.

FIG. 2B shows that the relative frequency of CD137⁺ CD8 cells of totalCD8+ TILs in is elevated in MC38 colon carcinoma tumors in animalstreated with anti-PD-1 antibodies (PD-1 group) when compared to vehicletreated (PBS) group. p=0.0475 vehicle vs anti-PD-1 antibody treatedgroups. Each point represents one mouse. Data are representative of atleast 2 independent experiments.

FIG. 3A shows that OX40 surface expression (gMFI) is elevated on TILs inMC38 colon carcinoma tumors in animals treated with anti-PD-1 antibodies(PD-1 group) when compared to vehicle treated (PBS) group. p=0.0013vehicle vs anti-PD-1 antibody treated groups. Each point represents onemouse. Data are representative of at least 2 independent experiments.

FIG. 3B shows that the relative frequency of OX40⁺ CD8 cells of totalCD8⁺ TILs in is elevated in MC38 colon carcinoma tumors in animalstreated with anti-PD-1 antibodies (PD-1 group) when compared to vehicletreated (PBS) group. p=0.03 vehicle vs anti-PD-1 antibody treatedgroups. Each point represents one mouse. Data are representative of atleast 2 independent experiments.

FIG. 4A shows that GITR surface expression (gMFI) is elevated on TILs inMC38 colon carcinoma tumors in animals treated with anti-PD-1 antibodies(PD-1 group) when compared to vehicle treated (PBS) group. p=0.0004vehicle vs anti-PD-1 antibody treated groups. Each point represents onemouse. Data are representative of at least 2 independent experiments.

FIG. 4B shows that the relative frequency of GITR⁺ CD8 cells of totalCD8⁺ TILs in is elevated in MC38 colon carcinoma tumors in animalstreated with anti-PD-1 antibodies (PD-1 group) when compared to vehicletreated (PBS) group. p=0.0015 vehicle vs anti-PD-1 antibody treatedgroups. Each point represents one mouse. Data are representative of atleast 2 independent experiments.

FIG. 5 shows that treatment with anti-TIM-3 antibodies after anti-PD-1antibody treatment further induces antigen-specific immune response. Theantibodies were tested in the CMV assay using PBMCs from CMV positivedonors, in which antigen-specific immune responses were induced withpp65 peptide pools. The cells were treated for 5 days with anti-PD-1antibody PD1B244, re-stimulated, and treated for 24 hours withanti-TIM-3 antibody TM3B105. Immune response was determined by measuringincreases in IFN-γ secretion. IgG2s Iso: IgG2sigma isotype control. CMV:sample treated with cytomegalovirus p65 peptides in the absence ofantibodies.

FIG. 6 shows the HCDR1 sequences of select anti-PD-1 antibodies and theHCDR1 genus sequence.

FIG. 7 shows the HCDR2 sequences of select anti-PD-1 antibodies and theHCDR2 genus sequence.

FIG. 8 shows the HCDR3 sequences of select anti-PD-1 antibodies and thefirst HCDR3 genus sequence.

FIG. 9 shows the HCDR3 sequences of select anti-PD-1 antibodies and thesecond HCDR3 genus sequence.

FIG. 10 shows the LCDR1 sequences of select anti-PD-1 antibodies and theLCDR1 genus sequence.

FIG. 11 shows the LCDR2 sequences of select anti-PD-1 antibodies and theLCDR2 genus sequence.

FIG. 12 shows the LCDR3 sequences of select anti-PD-1 antibodies and theLCDR3 genus sequence.

FIG. 13 shows the HCDR1 sequences of select anti-TIM-3 antibodies andthe HCDR1 genus sequence. The genus sequence was determined bygenerating molecular models for all Fv (VH/VL pairs) in MOE (CCG,Montreal) using a default protocol for antibody modeling. For CDRs thathave different lengths, these structural models were aligned based uponthe structurally conserved regions and the structurally equivalent CDRspositions were identified.

FIG. 14 shows the HCDR2 sequences of select anti-TIM-3 antibodies andthe HCDR2 genus sequence. The HCDR2 genus sequence was generated asdescribed for FIG. 10.

FIG. 15 shows the HCDR3 sequences of select anti-TIM-3 antibodies andthe first HCDR3 genus sequence. The HCDR3 genus sequence was generatedas described for FIG. 10.

FIG. 16 shows the LCDR1 sequences of select anti-TIM-3 antibodies andthe LCDR1 genus sequence. The LCDR1 genus sequence was generated asdescribed for FIG. 10.

FIG. 17 shows the LCDR2 sequences of select anti-TIM-3 antibodies andthe LCDR2 genus sequence. The LCDR2 genus sequence was generated asdescribed for FIG. 10.

FIG. 18 shows the LCDR3 sequences of select anti-TIM-3 antibodies andthe LCDR3 genus sequence. The LCDR3 genus sequence was generated asdescribed for FIG. 10.

FIG. 19A shows that TIGIT surface expression (gMFI) is elevated on TILsin MC38 colon carcinoma tumors in animals treated with anti-TIM-3antibodies (TIM-3 group) when compared to vehicle treated (PBS) group.p=0.0181 vehicle vs anti-TIM-3 antibody treated groups. Each pointrepresents one mouse. Data are representative of at least 2 independentexperiments.

FIG. 19B shows that the relative frequency of TIGIT+ CD8 cells of totalCD8+ TILs in is elevated in MC38 colon carcinoma tumors in animalstreated with anti-TIM-3 antibodies (TIM-3 group) when compared tovehicle treated (PBS) group. p=0.0475 vehicle vs anti-TIM-3 antibodytreated groups. Each point represents one mouse. Data are representativeof at least 2 independent experiments.

FIG. 20A shows that TIGIT surface expression (gMFI) is elevated on TILsin CT26 colon carcinoma tumors in animals treated with anti-TIM-3antibodies (TIM-3 group) when compared to vehicle treated (PBS) group.p<0.001 vehicle vs anti-TIM-3 antibody treated groups. Each pointrepresents one mouse. Data are representative of at least 2 independentexperiments.

FIG. 20B shows that the relative frequency of TIGIT+CD8 cells of totalCD8+ TILs in is elevated in CT26 colon carcinoma tumors in animalstreated with anti-TIM-3 antibodies (TIM-3 group) when compared tovehicle treated (PBS) group. p=0.0105 vehicle vs anti-TIM-3 antibodytreated groups. Each point represents one mouse. Data are representativeof at least 2 independent experiments.

FIG. 21 shows upregulation of TIM-3 expression on peripheral T cells inmelanoma patients PBMCs from treatment naïve melanoma patientsstimulated with melanoma antigen peptide pools (NY-ESO, gp100, MART-1)in the presence or absence of anti-PD-1 or anti-TIM-3 function blockingantibodies. Expression of TIM-3 was determined by flow cytometry onrestimulated cells on day 6.

FIG. 22A shows that TM3B403 treatment increases frequency of activatedNK cells in IL-2 stimulated human PBMCs. IgG2s: Isotype control. NK cellactivation was assessed as percentage (%) of CD69 expressing cells inthe stimulated PBMCs.

FIG. 22B shows that TM3B403 treatment increases frequency of activatedNK cells in IL-2 stimulated human PBMCs. IgG2s: Isotype control. NK cellactivation was assessed as percentage (%) of CD25 expressing cells inthe stimulated PBMCs.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though fully set forth.

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which the invention pertains.

Although any methods and materials similar or equivalent to thosedescribed herein may be used in the practice for testing of the presentinvention, exemplary materials and methods are described herein. Indescribing and claiming the present invention, the following terminologywill be used.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

“Specific binding” or “specifically binds” or “binds” refers to anantibody binding to an antigen or an epitope within the antigen withgreater affinity than for other antigens. Typically, the antibody bindsto the antigen or the epitope within the antigen with an equilibriumdissociation constant (K_(D)) of about 1×10⁻⁸ M or less, for exampleabout 1×10⁻⁹ M or less, about 1×10⁻¹⁰ M or less, about 1×10⁻¹¹ M orless, or about 1×10⁻¹² M or less, typically with the K_(D) that is atleast one hundred fold less than its K_(D) for binding to a non-specificantigen (e.g., BSA, casein). The dissociation constant may be measuredusing standard procedures. Antibodies that specifically bind to theantigen or the epitope within the antigen may, however, havecross-reactivity to other related antigens, for example to the sameantigen from other species (homologs), such as human or monkey, forexample Macaca fascicularis (cynomolgus, cyno), Pan troglodytes(chimpanzee, chimp) or Callithrix jacchus (common marmoset, marmoset).While a monospecific antibody specifically binds one antigen or oneepitope, a bispecific antibody specifically binds two distinct antigensor two distinct epitopes.

“Antibodies” is meant in a broad sense and includes immunoglobulinmolecules including monoclonal antibodies including murine, human,humanized and chimeric monoclonal antibodies, antigen-binding fragments,bispecific or multispecific antibodies, dimeric, tetrameric ormultimeric antibodies, single chain antibodies, domain antibodies andany other modified configuration of the immunoglobulin molecule thatcomprises an antigen binding site of the required specificity. “Fulllength antibodies” are comprised of two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds as well as multimersthereof (for example IgM). Each heavy chain is comprised of a heavychain variable region (VH) and a heavy chain constant region (comprisedof domains CH1, hinge CH2 and CH3). Each light chain is comprised of alight chain variable region (VL) and a light chain constant region (CL).The VH and the VL regions may be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with framework regions (FR). Each VH and VL is composed ofthree CDRs and four FR segments, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4.

“Complementarity determining regions (CDR)” are “antigen binding sites”in an antibody. CDRs may be defined using various terms: (i)Complementarity Determining Regions (CDRs), three in the VH (HCDR1,HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3) are based onsequence variability (Wu and Kabat, (1970) J Exp Med 132:211-50; Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991).(ii) “Hypervariable regions”, “HVR”, or “HV”, three in the VH (H1, H2,H3) and three in the VL (L1, L2, L3) refer to the regions of an antibodyvariable domains which are hypervariable in structure as defined byChothia and Lesk (Chothia and Lesk, (1987) Mol Biol 196:901-17). TheInternational ImMunoGeneTics (IMGT) database (http://www_imgt_org)provides a standardized numbering and definition of antigen-bindingsites. The correspondence between CDRs, HVs and IMGT delineations isdescribed in Lefranc et al., (2003) Dev Comparat Immunol 27:55-77. Theterm “CDR”, “HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCDR2” and “LCDR3” asused herein includes CDRs defined by any of the methods described supra,Kabat, Chothia or IMGT, unless otherwise explicitly stated in thespecification.

Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE,IgG and IgM, depending on the heavy chain constant domain amino acidsequence. IgA and IgG are further sub-classified as the isotypes IgA1,IgA2, IgG1, IgG2, IgG3 and IgG4. Antibody light chains of any vertebratespecies may assigned to one of two clearly distinct types, namely kappa(κ) and lambda (λ), based on the amino acid sequences of their constantdomains.

“Antibody fragments” or “antigen-binding portion” refers to a portion ofan immunoglobulin molecule that retains the antigen binding propertiesof the parental full length antibody. Exemplary antigen-binding portionsare heavy chain complementarity determining regions (HCDR) 1, 2 and 3,light chain complementarity determining regions (LCDR) 1, 2 and 3, aheavy chain variable region (VH), a light chain variable region (VL),Fab, F(ab′)2, Fd and Fv fragments as well as domain antibodies (dAb)consisting of either one VH or VL domain. VH and VL domains may belinked together via a synthetic linker to form various types of singlechain antibody designs where the VH/VL domains may pairintramolecularly, or intermolecularly in those cases when the VH and VLdomains are expressed by separate single chain antibody constructs, toform a monovalent antigen binding site, such as single chain Fv (scFv)or diabody; described for example in Int. Patent Publ. Nos.WO1998/44001, WO1988/01649, WO1994/13804 and WO1992/01047.

“Monoclonal antibody” refers to an antibody population with single aminoacid composition in each heavy and each light chain, except for possiblewell known alterations such as removal of C-terminal lysine from theantibody heavy chain. Monoclonal antibodies typically bind one antigenicepitope, except that multispecific monoclonal antibodies bind two ormore distinct antigens or epitopes. Bispecific monoclonal antibodiesbind two distinct antigenic epitopes. Monoclonal antibodies may haveheterogeneous glycosylation within the antibody population. Monoclonalantibodies may be monospecific or multispecific, or monovalent, bivalentor multivalent. A multispecific antibody, such as a bispecific antibodyor a trispecific antibody is included in the term monoclonal antibody.

“Isolated antibody” refers to an antibody or antibody fragment that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody specifically binding PD-1 issubstantially free of antibodies that specifically bind antigens otherthan PD-1). An isolated antibody specifically binding TIM-3 issubstantially free of antibodies that specifically bind antigens otherthan TIM-3. In case of bispecific PD-1/TIM-3 antibodies, the bispecificantibody specifically binds both PD-1 and TIM-3, and is substantiallyfree of antibodies that specifically bind antigens other that PD-1 andTIM-3. “Isolated antibody” encompasses antibodies that are isolated to ahigher purity, such as antibodies that are 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% pure.

“Humanized antibodies” refers to antibodies in which at least one CDR isderived from non-human species and the variable region frameworks arederived from human immunoglobulin sequences. Humanized antibodies mayinclude intentionally introduced mutations in the framework regions sothat the framework may not be an exact copy of expressed humanimmunoglobulin or germline gene sequences.

“Human antibody” refers to an antibody having heavy and light chainvariable regions in which both the framework and all 6 CDRs are derivedfrom sequences of human origin. If the antibody contains a constantregion or a portion of the constant region, the constant region also isderived from sequences of human origin.

Human antibody comprises heavy or light chain variable regions that are“derived from” sequences of human origin if the variable regions of theantibody are obtained from a system that uses human germlineimmunoglobulin or rearranged immunoglobulin genes. Such exemplarysystems are human immunoglobulin gene libraries displayed on phage, andtransgenic non-human animals such as mice or rats carrying humanimmunoglobulin loci as described herein. “Human antibody” may containamino acid differences when compared to the human germlineimmunoglobulin or rearranged immunoglobulin genes due to for examplenaturally occurring somatic mutations or intentional introduction ofsubstitutions into the framework or antigen binding site, or both.Typically, “human antibody” is at least about 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identical in amino acid sequence to an amino acid sequenceencoded by human germline immunoglobulin or rearranged immunoglobulingenes. In some cases, “human antibody” may contain consensus frameworksequences derived from human framework sequence analyses, for example asdescribed in Knappik et al., (2000) J Mol Biol 296:57-86, or syntheticHCDR3 incorporated into human immunoglobulin gene libraries displayed onphage, for example as described in Shi et al., (2010) J Mol Biol397:385-96, and in Int. Patent Publ. No. WO2009/085462.

Human antibodies derived from human immunoglobulin sequences may begenerated using systems such as phage display incorporating syntheticCDRs and/or synthetic frameworks, or may be subjected to in vitromutagenesis to improve antibody properties, resulting in antibodies thatare not expressed by the human antibody germline repertoire in vivo.

“Recombinant” refers to antibodies and other proteins that are prepared,expressed, created or isolated by recombinant means.

“Epitope” refers to a portion of an antigen to which an antibodyspecifically binds. Epitopes typically consist of chemically active(such as polar, non-polar or hydrophobic) surface groupings of moietiessuch as amino acids or polysaccharide side chains and may have specificthree-dimensional structural characteristics, as well as specific chargecharacteristics. An epitope may be composed of contiguous and/ordiscontiguous amino acids that form a conformational spatial unit. For adiscontiguous epitope, amino acids from differing portions of the linearsequence of the antigen come in close proximity in 3-dimensional spacethrough the folding of the protein molecule. Antibody “epitope” dependson the methodology used to identify the epitope.

“Multispecific” refers to an antibody that specifically binds at leasttwo distinct antigens or two distinct epitopes within the antigens, forexample three, four or five distinct antigens or epitopes.

“Bispecific” refers to an antibody that specifically binds two distinctantigens or two distinct epitopes within the same antigen. Thebispecific antibody may have cross-reactivity to other related antigens,for example to the same antigen from other species (homologs), such ashuman or monkey, for example Macaca fascicularis (cynomolgus, cyno), Pantroglodytes (chimpanzee, chimp) or Callithrix jacchus (common marmoset,marmoset), or may bind an epitope that is shared between two or moredistinct antigens.

“Variant” refers to a polypeptide or a polynucleotide that differs froma reference polypeptide or a reference polynucleotide by one or moremodifications for example, substitutions, insertions or deletions.

“Vector” refers to a polynucleotide capable of being duplicated within abiological system or that can be moved between such systems. Vectorpolynucleotides typically contain elements, such as origins ofreplication, polyadenylation signal or selection markers, that functionto facilitate the duplication or maintenance of these polynucleotides ina biological system. Examples of such biological systems may include acell, virus, animal, plant, and reconstituted biological systemsutilizing biological components capable of duplicating a vector. Thepolynucleotide comprising a vector may be DNA or RNA molecules or ahybrid of these.

“Expression vector” refers to a vector that can be utilized in abiological system or in a reconstituted biological system to direct thetranslation of a polypeptide encoded by a polynucleotide sequencepresent in the expression vector.

“Polynucleotide” refers to a synthetic molecule comprising a chain ofnucleotides covalently linked by a sugar-phosphate backbone or otherequivalent covalent chemistry. cDNA is a typical example of apolynucleotide.

“Polypeptide” or “protein” refers to a molecule that comprises at leasttwo amino acid residues linked by a peptide bond to form a polypeptide.Small polypeptides of less than 50 amino acids may be referred to as“peptides”.

PD-1 refers to human programmed cell death protein 1, PD-1. PD-1 is alsoknown as CD279 or PDCD1. The amino acid sequence of the mature humanPD-1 (without signal sequence) is shown in SEQ ID NO: 1. Theextracellular domain spans residues 1-150, the transmembrane domainspans residues 151-171 and the cytoplasmic domain spans residues 172-268of SEQ ID NO: 1. Throughout the specification, “the extracellular domainof human PD-1 huPD1-ECD” refers to protein having amino acid sequence ofresidues 1-149 of SEQ ID NO: 1, and shown in SEQ ID NO:2. “PD-1” in thespecification refers to human mature PD-1, unless explicitly stated tothe contrary.

TIM-3 refers to human hepatitis A virus cellular receptor 2, also calledHAVCR2. The amino acid sequence of the mature human TIM-3 (withoutsignal sequence) is shown in SEQ ID NO: 138. The extracellular domainspans residues 1-181, the transmembrane domain spans residues 182-202and the cytoplasmic domain spans residues 203-280 of SEQ ID NO: 138.Throughout the specification, “the extracellular domain of human TIM-3huTIM-3-ECD” refers to protein having amino acid sequence of residues1-179 of SEQ ID NO: 138, and shown in SEQ ID NO: 89. TIM-3 in thespecification refers to human mature TIM-3, unless explicitly stated tothe contrary.

“In combination with” means that two or more therapeutics areadministered to a subject together in a mixture, concurrently as singleagents or sequentially as single agents in any order.

“Overexpress”, “overexpressed” and “overexpressing” is usedinterchangeably and refers to a sample such as a cancer cell, malignantcell or cancer tissue that has measurably higher levels of PD-1, TIM-3,PD-L1, PD-L2 or TIM-3 ligand when compared to a reference sample. Theoverexpression may be caused by gene amplification or by increasedtranscription or translation. Expression and overexpression of proteinin the sample may be measured using well know assays using for exampleELISA, immunofluorescence, flow cytometry or radioimmunoassay on live orlysed cells. Expression and overexpression of a polynucleotide in thesample may be measured for example using fluorescent in situhybridization, Southern blotting, or PCR techniques. A protein or apolynucleotide is overexpressed when the level of the protein or thepolynucleotide in the sample at least 1.5-fold higher or statisticallysignificant when compared to the reference sample. Selection of thereference sample is known.

“Sample” refers to a collection of similar fluids, cells, or tissuesisolated from a subject, as well as fluids, cells, or tissues presentwithin a subject. Exemplary samples are biological fluids such as blood,serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid,tear drops, feces, sputum, mucosal secretions of the secretory tissuesand organs, vaginal secretions, ascites fluids such as those associatedwith non-solid tumors, fluids of the pleural, pericardial, peritoneal,abdominal and other body cavities, fluids collected by bronchial lavage,liquid solutions contacted with a subject or biological source, forexample, cell and organ culture medium including cell or organconditioned medium, lavage fluids and the like, tissue biopsies, fineneedle aspirations or surgically resected tumor tissue.

A “cancer cell” or a “tumor cell” refers to a cancerous, pre-cancerousor transformed cell, either in vivo, ex vivo, or in tissue culture, thathas spontaneous or induced phenotypic changes. These changes do notnecessarily involve the uptake of new genetic material. Althoughtransformation may arise from infection with a transforming virus andincorporation of new genomic nucleic acid, uptake of exogenous nucleicacid or it can also arise spontaneously or following exposure to acarcinogen, thereby mutating an endogenous gene. Transformation/canceris exemplified by morphological changes, immortalization of cells,aberrant growth control, foci formation, proliferation, malignancy,modulation of tumor specific marker levels, invasiveness, tumor growthin suitable animal hosts such as nude mice, and the like, in vitro, invivo, and ex vivo (Freshney, Culture of Animal Cells: A Manual of BasicTechnique (3rd ed. 1994)).

“About” means within an acceptable error range for the particular valueas determined by one of ordinary skill in the art, which will depend inpart on how the value is measured or determined, i.e., the limitationsof the measurement system. Unless explicitly stated otherwise within theExamples or elsewhere in the Specification in the context of aparticular assay, result or embodiment, “about” means within onestandard deviation per the practice in the art, or a range of up to 5%,whichever is larger.

“Bispecific PD-1/TIM-3 antibody”, “PD-1/TIM-3 antibody”, “bispecificanti-PD-1/TIM-3 antibody” or “anti-PD-1/TIM-3 antibody” refers to amolecule comprising at least one binding domain specifically bindingPD-1 and at least one binding domain specifically binding TIM-3. Thedomains specifically binding PD-1 and TIM-3 are typically VH/VL pairs.The bispecific anti-PD-1/TIM-3 antibody may be monovalent in terms ofits binding to either PD-1 or TIM-3.

“Valent” refers to the presence of a specified number of binding sitesspecific for an antigen in a molecule. As such, the terms “monovalent”,“bivalent”, “tetravalent”, and “hexavalent” refer to the presence ofone, two, four and six binding sites, respectively, specific for anantigen in a molecule.

“An antigen specific CD4⁺ or CD8⁺ T cell” refers to a CD4⁺ or CD8⁺ Tcell activated by a specific antigen, or immunostimulatory epitopethereof.

“CD137” (also called tumor necrosis factor receptor superfamily member9, TNFRSF9, 4-1BBL) refers to a human CD137 molecule having the aminoacid sequence shown in SEQ ID NO: 281.

SEQ ID NO: 281 MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCEL

“TIGIT” (also called T-cell immunoreceptor with Ig and ITIM domains)refers to human TIGIT molecule having the amino acid sequence shown inSEQ ID NO: 301.

SEQ ID NO: 301 MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG

“Agonist” refers to a molecule that, when bound to a cellular protein,induces at least one reaction or activity that is induced by a naturalligand of the protein. The molecule is an agonist when the at least onereaction or activity is induced by at least about 30%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% greater than the atleast one reaction or activity induced in the absence of the agonist(e.g., negative control), or when the induction is statisticallysignificant when compared to the induction in the absence of theagonist. Agonist may be an antibody, a soluble ligand, or a smallmolecule. An exemplary agonist is an agonistic antibody thatspecifically binds a T cell activating molecule.

“Antagonist” refers to a molecule that, when bound to a cellularprotein, suppresses at least one reaction or activity that is induced bya natural ligand of the protein. A molecule is an antagonist when the atleast one reaction or activity is suppressed by at least about 30%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more thanthe at least one reaction or activity suppressed in the absence of theantagonist (e.g., negative control), or when the suppression isstatistically significant when compared to the suppression in theabsence of the antagonist. Antagonist may be an antibody, a solubleligand, a small molecule, a DNA or RNA such as siRNA. Exemplaryantagonists are an antagonistic antibody specifically binding PD-1, anantagonistic antibody specifically binding TIM-3, an antagonisticbispecific PD-1/TIM-3 antibody or an antagonistic antibody specificallybinding a T cell inhibitory molecule. A typical reaction or activitythat is induced by PD-1 binding to its receptor PD-L1 or PD-L2 may bereduced antigen-specific CD4⁺ or CD8⁺ cell proliferation or reducedinterferon-γ (IFN-γ) production by T cells, resulting in suppression ofimmune responses against for example tumor. A typical reaction oractivity that is induced by TIM-3 binding to its receptor, such asgalectin-9, may be reduced antigen specific CD4⁺ or CD8⁺ cellproliferation, reduced IFN-γ production by T cells, or reduced CD137surface expression on CD4⁺ or CD8⁺ cells, resulting in suppression ofimmune responses against for example tumor. Similarly, a typicalreaction or activity that is induced by a T cell inhibitory molecule isimmunosuppression. Hence, an antagonistic PD-1 antibody specificallybinding PD-1, an antagonistic antibody specifically binding TIM-3, anantagonistic bispecific PD-1/TIM-3 antibody, or an antagonistic antibodyspecifically binding a T cell inhibitory molecule induces immuneresponses by inhibiting the inhibitory pathways.

“Subject” includes any human or nonhuman animal “Nonhuman animal”includes all vertebrates, e.g., mammals and non-mammals, such asnonhuman primates, sheep, dogs, cats, horses, cows chickens, amphibians,reptiles, etc. Except when noted, the terms “patient” or “subject” areused interchangeably.

The numbering of amino acid residues in the antibody constant regionthroughout the specification is according to the EU index as describedin Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991), unless otherwise explicitly stated.

Conventional one and three-letter amino acid codes are used herein asshown in Table 1.

TABLE 1 Amino acid Three-letter code One-letter code Alanine Ala AArginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys C GlutamateGln E Glutamine Glu Q Glycine Gly G Histidine His H Isoleucine Ile ILysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P SerineSer S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val VCompositions of Matter

The present invention provides antagonistic antibodies specificallybinding PD-1, antagonistic antibodies specifically binding TIM-3, andantagonistic bispecific PD-1/TIM-3 antibodies. The present inventionprovides polypeptides and polynucleotides encoding the antibodies of theinvention or complementary nucleic acids thereof, vectors, host cells,and methods of making and using them.

Antagonistic Antibodies Specifically Binding PD-1

PD-1, upon ligand engagement, suppresses T cell functions throughmultiple mechanisms (Pauken & Wherry (2015) Trends in Immunology 36(4):265-276). PD-1 engagement directly inhibits T cell receptor (TCR)signaling through co-localization with the TCR and subsequent inductionof dephosphorylation of TCR proximal signaling molecules, inhibition ofRas/MEK/ERK pathway leading to inhibition of the cell cycle progressionand T cell proliferation, inhibition of cell growth and survival andreprogramming of T cell metabolism through suppression of PI3K/AKTpathway, leading to the upregulation of the BATF transcription factor,and modulation of development, maintenance and function of regulatory Tcells. PD-1 has also been proposed to increase T cell motility and tolimit duration of interaction between T cells and target cells, therebyreducing the extent of T cell activation (Honda et al., (2014) Immunity40(2):235-47).

Tumors have co-opted the PD-1 pathway to downregulate T cell function inthe tumor microenvironment (TME) and to evade immune destruction. In theTME, under conditions of persistent antigen and inflammation, T cellsbecome exhausted, or dysfunctional, and progressively lose theireffector function and proliferative capacity. Exhausted T cells expresshigh levels of PD-1, often together with other inhibitory receptors suchas TIM-3 or LAG-3 (Pauken & Wherry (2015) Trends in Immunology 36(4):265-276). One of the PD-1 ligands, PD-L1, is also upregulated in varioustumors. PD-L1 expression occurs on the cancer cells themselves and/orinfiltrating immune cells, including tumor associated macrophages,dendritic cells, fibroblasts and activated T cells (Chen et al., 2012Clin Cancer Res 18(24):6580-7). In this setting, PD-1 engagement ishypothesized to limit anti-tumor T cell responses and lead to immuneevasion. Recent studies have shown that a higher frequency and level ofPD-1 expression occurs on tumor infiltrating lymphocytes (TILs) inmultiple solid tumors Importantly, these PD-1⁺ TILs are functionallyimpaired, as evidenced by lower proliferation and effector functions(Pauken & Wherry; 2015, Trends in Immunology 36(4): 265-276) These datasupport the hypothesis that PD-1 mediates immune suppression in the TME.

T cell exhaustion in tumors is reversible, at least partially, by PD-1pathway blockade. Anti-PD-1/PD-L1 antibodies have been shown to enhanceT cell function and lead to improved anti-tumor immunity in a number ofpreclinical tumor models. PD-1/PD-L1 antibodies have also shownencouraging clinical responses in multiple solid tumors, with 20-40%overall response rate (ORR) in melanoma, 10-24% in non-small cell lungcancer (NSCLC), 12-31% in renal cell carcinoma (RCC), 24-52% in bladdercancer, and 20% in head and neck cancer (Swaika et al., (2015) MolImmunol 67(2 Pt A):4-17).

The invention provides an isolated antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof comprising a heavychain complementarity determining region 1 (HCDR1), a HCDR2 and a HCDR3of SEQ ID NOs: 82, 83 and 84, respectively, or SEQ ID NOs: 82, 83 and85, respectively.

The invention also provides an isolated antagonistic antibodyspecifically binding PD-1 or an antigen-binding portion thereofcomprising a light chain complementarity determining region 1 (LCDR1), aLCDR2 and a LCDR3 of SEQ ID NOs: 86, 87 and 88, respectively.

The invention also provides an isolated antagonistic antibodyspecifically binding PD-1 or an antigen-binding portion thereofcomprising the HCDR1, the HCDR2 and the HCDR3 of SEQ ID NOs: 82, 83 and84, respectively, and the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:86, 87 and 88, respectively.

The invention also provides an isolated antagonistic antibodyspecifically binding PD-1 or an antigen-binding portion thereofcomprising the HCDR1, the HCDR2 and the HCDR3 of SEQ ID NOs: 82, 83 and85, respectively, and the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:86, 87 and 88, respectively.

SEQ ID NOs: 82, 83, 84, 85, 86, 87 and 88 represent the HCDR1, theHCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 genus sequences ofaffinity-matured variants of antagonistic antibodies specificallybinding PD-1 having similar HCDR1, HCDR2, LCDR1, LCDR2 and LCDR3sequences, and two similar HCDR3 groups of sequences. Antibodies withinthe genus bind PD-1 with the K_(D) of less than about 1×10⁻⁷ M, such asless than about 1×10⁻⁸ M, for example less than about 1×10⁻⁹ M, or forexample less than about 1×10⁻¹° M. Exemplary such antibodies areantibodies having the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2and the LCDR3 amino acid sequences of antibodies PD1B114, PD1B149,PD1B160, PD1B162, PD1B164, PD1B11, PD1B183, PD1B184, PD1B185, PD1B187,PD1B71, PD1B177, PD1B70, PD1B175, PD1B194, PD1B195, PD1B196, PD1B197,PD1B198, PD1B199, PD1B200, PD1B201 and PD1B244 as described herein.

SEQ ID NO: 82 X₁YX₂IX₃,whereinX₁ is S or D;X₂ is V or A; andX₃ is H or S.

SEQ ID NO: 83 GIIPIX₄X₅TANYAQKFQG,whereinX₄ is Y or F; andX₅ is G or D.

SEQ ID NO: 84 PGLAAAYDTGX₆LDY,whereinX₆ is N or S.

SEQ ID NO: 85 GX₇X₈X₉X₁₀TGX₁₁LDY,whereinX₇ is T or Y;X₈ is L or V;X₉ is D or R;X₁₀ is R or A; andX₁₁ is H or M.

SEQ ID NO: 86 RASQSVX₁₂X₁₃YLA,whereinX₁₂ is S, R or D; andX₁₃ is S or N.

SEQ ID NO: 87 DASX₁₄RAT,whereinX₁₄ is N, D, Y, S or T.

SEQ ID NO: 88 QQRX₁₅X₁₆WPLT,whereinX₁₅ is S, N, G, E, D, W or A; andX₁₆ is N, Y, E or A.

In some embodiments, the isolated antagonistic antibody specificallybinding PD-1 or the antigen-binding portion thereof has one, two, three,four or five of the following properties:

-   -   a) enhances an activation of antigen specific CD4⁺ or CD8⁺ T        cells in a dose dependent manner, wherein the activation is        measured using a cytomegalovirus antigen recall assay (CMV        assay) as described in Example 1;    -   b) binds human PD-1 with an equilibrium dissociation constant        (K_(D)) of less than about 100 nM, wherein the K_(D) is measured        using ProteOn XPR36 system at +25° C.;    -   c) binds human PD-1 with the K_(D) of less than about 1 nM,        wherein the K_(D) is measured using ProteOn XPR36 system at +25°        C.;    -   d) binds cynomolgus PD-1 with the K_(D) of less than about 100        nM, wherein the K_(D) is measured using ProteOn XPR36 system at        +25° C., or    -   e) binds cynomolgus PD-1 with the K_(D) of less than about 1 nM,        wherein the K_(D) is measured using ProteOn XPR36 system at +25°        C.

Exemplary such antibodies are PD-1 antibodies PD1B114, PD1B149, PD1B160,PD1B162, PD1B164, PD1B11, PD1B183, PD1B184, PD1B185, PD1B187, PD1B71,PD1B177, PD1B70, PD1B175, PD1B194, PD1B195, PD1B196, PD1B197, PD1B198,PD1B199, PD1B200, PD1B201 and PD1B244 as described herein.

In some embodiments, the isolated antagonistic antibody specificallybinding PD-1 or the antigen-binding portion thereof enhances anactivation of antigen specific CD4⁺ or CD8⁺ T cells in a dose dependentmanner, wherein the activation is measured using a cytomegalovirusantigen recall assay (CMV assay) as described in Example 1, and bindshuman PD-1 with an equilibrium dissociation constant (K_(D)) of lessthan about 100 nM, wherein the K_(D) is measured using ProteOn XPR36system at +25° C.

In some embodiments, the isolated antagonistic antibody specificallybinding PD-1 or the antigen-binding portion thereof enhances anactivation of antigen specific CD4⁺ or CD8⁺ T cells in dose dependentmanner, wherein the activation is measured using a cytomegalovirusantigen recall assay (CMV assay) as described in Example 1, and bindshuman PD-1 with an equilibrium dissociation constant (K_(D)) of lessthan about 10 nM, wherein the K_(D) is measured using ProteOn XPR36system at +25° C.

In some embodiments, the isolated antagonistic antibody specificallybinding PD-1 or the antigen-binding portion thereof enhances anactivation of antigen specific CD4⁺ or CD8⁺ T cells in dose dependentmanner, wherein the activation is measured using a cytomegalovirusantigen recall assay (CMV assay) as described in Example 1, and bindscynomolgus PD-1 with an equilibrium dissociation constant (K_(D)) ofless than about 100 nM, wherein the K_(D) is measured using ProteOnXPR36 system at +25° C.

In some embodiments, the isolated antagonistic antibody specificallybinding PD-1 or the antigen-binding portion thereof enhances anactivation of antigen specific CD4⁺ or CD8⁺ T cells in dose dependentmanner, wherein the activation is measured using a cytomegalovirusantigen recall assay (CMV assay) as described in Example 1, and bindscynomolgus PD-1 with an equilibrium dissociation constant (K_(D)) ofless than about 10 nM, wherein the K_(D) is measured using ProteOn XPR36system at +25° C.

Activation of antigen specific CD4⁺ or CD8⁺ T cells may be assessed bymeasuring increased T cell proliferation in a Mixed Lymphocyte Reaction(MLR) assay, increased interferon-γ (IFN-γ) secretion in the MLR assay,increased TNF-α secretion in the MLR assay, increased IFN-γ secretion ina cytomegalovirus antigen assay (CMV assay) or increased TNF-α secretionin the CMV assay using known protocols and those described in Example 1.Antibodies of the invention enhance the activation of antigen specificCD4⁺ or CD8⁺ T when the measured T cell functionality is increased bythe antibodies of the invention in a dose-dependent manner.

The affinity of an antibody to human or cynomolgus PD-1 may bedetermined experimentally using any suitable method. Such methods mayutilize ProteOn XPR36, Biacore 3000 or KinExA instrumentation, ELISA orcompetitive binding assays known to those skilled in the art. Themeasured affinity of a particular antibody/PD-1 interaction may vary ifmeasured under different conditions (e.g., osmolarity, pH). Thus,measurements of affinity and other binding parameters (e.g., K_(D),K_(on), K_(off)) are typically made with standardized conditions and astandardized buffer, such as the buffer described herein. Skilled in theart will appreciate that the internal error for affinity measurementsfor example using Biacore 3000 or ProteOn (measured as standarddeviation, SD) may typically be within 5-33% for measurements within thetypical limits of detection. Therefore the term “about” in the contextof K_(D) reflects the typical standard deviation in the assay. Forexample, the typical SD for a K_(D) of 1×10⁻⁹M is up to +0.33×10⁻⁹M.

In some embodiments, the antagonistic antibody specifically binding PD-1or the antigen-binding portion thereof comprises the HCDR1, the HCDR2and the HCDR3 contained within a heavy chain variable region (VH) of SEQID NOs: 41, 42, 43, 44, 45, 46, 47 or 48, wherein the HCDR1, the HCDR2and the HCDR3 are defined by Chothia, Kabat, or IMGT.

In some embodiments, the antagonistic antibody specifically binding PD-1or the antigen-binding portion thereof of the invention comprises theLCDR1, the LCDR2 and the LCDR3 contained within a light chain variableregion (VL) of SEQ ID NOs: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61 or 62, wherein the LCDR1, the LCDR2 and the LCDR are defined byChothia, Kabat, or IMGT.

In some embodiments, the antagonistic antibody specifically binding PD-1or the antigen-binding portion thereof of the invention comprises

the HCDR1 of SEQ ID NOs: 10, 11 or 12;

the HCDR2 of SEQ ID NOs: 13, 14 or 15; and

the HCDR3 of SEQ ID NOs: 16, 17, 18 or 19.

In some embodiments, the antagonistic antibody specifically binding PD-1or the antigen-binding portion thereof of the invention comprises

the LCDR1 of SEQ ID NOs: 20, 21, 22, 23, 24 or 25;

the LCDR2 of SEQ ID NOs: 26, 27, 28, 29 or 30; and

the LCDR3 of SEQ ID NOs: 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

In some embodiments, the antagonistic antibody specifically binding PD-1or the antigen-binding portion thereof of the invention comprises

the HCDR1 of SEQ ID NOs: 10, 11 or 12;

the HCDR2 of SEQ ID NOs: 13, 14 or 15;

the HCDR3 of SEQ ID NOs: 16, 17, 18 or 19;

the LCDR1 of SEQ ID NOs: 20, 21, 22, 23, 24 or 25;

the LCDR2 of SEQ ID NOs: 26, 27, 28, 29 or 30; and

the LCDR3 of SEQ ID NOs: 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40.

In some embodiments, the antagonistic antibody specifically binding PD-1or the antigen-binding portion thereof of the invention comprises theHCDR1, the HCDR2 and the HCDR3 of

SEQ ID NOs: 10, 13 and 16, respectively;

SEQ ID NOs: 10, 14 and 16, respectively;

SEQ ID NOs: 10, 13 and 17, respectively;

SEQ ID NOs: 10, 13 and 18, respectively;

SEQ ID NOs: 11, 15 and 18, respectively;

SEQ ID NOs: 10, 13 and 19, respectively;

SEQ ID NOs: 10, 14 and 17, respectively; or

SEQ ID NOs: 12, 13 and 19, respectively.

In some embodiments, the antagonistic antibody specifically binding PD-1or the antigen-binding portion thereof of the invention comprises theLCDR1, the LCDR2 and the LCDR3 of

SEQ ID NOs: 20, 26 and 31, respectively;

SEQ ID NOs: 21, 26 and 32, respectively;

SEQ ID NOs: 22, 27 and 33, respectively;

SEQ ID NOs: 22, 26 and 34, respectively;

SEQ ID NOs: 23, 28 and 35, respectively;

SEQ ID NOs: 20, 26 and 36, respectively;

SEQ ID NOs: 21, 27 and 37, respectively;

SEQ ID NOs: 23, 26 and 32, respectively;

SEQ ID NOs: 22, 26 and 32, respectively;

SEQ ID NOs: 24, 26 and 38, respectively;

SEQ ID NOs: 20, 29 and 39, respectively;

SEQ ID NOs: 20, 30 and 32, respectively;

SEQ ID NOs: 25, 26 and 40, respectively; or

SEQ ID NOs: 24, 26 and 32, respectively.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof comprising the HCDR1,the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:10, 14, 17, 23, 26 and 32, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofbinds human PD-1 with an equilibrium dissociation constant (K_(D)) ofless than about 100 nM, optionally less than about 10 nM, for exampleless than about 1 nM such as less than about 500 pM, wherein the K_(D)is measured using ProteOn XPR36 system at +25° C.

In some embodiments, the antibody or the antigen-binding portion thereofbinds cynomolgous PD-1 with an equilibrium dissociation constant (K_(D))of less than about 100 nM, optionally less than about 10 nM, for exampleless than about 1 nM such as less than about 500 pM, wherein the K_(D)is measured using ProteOn XPR36 system at +25° C.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 48 and the VL of SEQ ID NO: 56.

In some embodiments, the VH and the VL are encoded by polynucleotidesequences of SEQ ID NOs: 196 and 197, respectively.

In some embodiments, the antibody is an IgG4 isotype, optionallycomprising a S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is an IgG4/K isotype, optionallycomprising the S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 48 andthe VL of SEQ ID NO: 56 and is an IgG4 isotype, optionally comprisingthe S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 48 andthe VL of SEQ ID NO: 56 and is an IgG4/K isotype comprising the S228Psubstitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ IDNO: 72 and a light chain (LC) of SEQ ID NO: 73.

In some embodiments, the antibody is an IgG2 isotype, optionallycomprising V234A, G237A, P238S, H268A, V309L, A330S and P331Ssubstitutions when compared to the wild type IgG2.

In some embodiments, the antibody is an IgG2/K isotype, optionallycomprising V234A, G237A, P238S, H268A, V309L, A330S and P331Ssubstitutions when compared to the wild type IgG2.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 48 andthe VL of SEQ ID NO: 56 and is an IgG2/K isotype, optionally comprisingV234A, G237A, P238S, H268A, V309L, A330S and P331S substitutions whencompared to the wild type IgG2.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 48 andthe VL of SEQ ID NO: 56 and is an IgG2/K isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treatingcancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The antibody is suitable for use in therapy, for example in treating amelanoma.

The antibody is suitable for use in therapy, for example in treating alung cancer.

The antibody is suitable for use in therapy, for example in treatingnon-small cell lung cancer (NSCLC).

The antibody is suitable for use in therapy, for example in treating asquamous NSCLC.

The antibody is suitable for use in therapy, for example in treating anon-squamous NSCLC.

The antibody is suitable for use in therapy, for example in treating alung adenocarcinoma.

The antibody is suitable for use in therapy, for example in treating arenal cell carcinoma (RCC).

The antibody is suitable for use in therapy, for example in treating amesothelioma.

The antibody is suitable for use in therapy, for example in treating anasopharyngeal carcinoma (NPC).

The antibody is suitable for use in therapy, for example in treating acolorectal cancer.

The antibody is suitable for use in therapy, for example in treating aprostate cancer.

The antibody is suitable for use in therapy, for example in treating acastration-resistant prostate cancer.

The antibody is suitable for use in therapy, for example in treating astomach cancer.

The antibody is suitable for use in therapy, for example in treating anovarian cancer.

The antibody is suitable for use in therapy, for example in treating agastric cancer.

The antibody is suitable for use in therapy, for example in treating aliver cancer.

The antibody is suitable for use in therapy, for example in treating apancreatic cancer.

The antibody is suitable for use in therapy, for example in treating athyroid cancer.

The antibody is suitable for use in therapy, for example in treating asquamous cell carcinoma of the head and neck.

The antibody is suitable for use in therapy, for example in treating acarcinomas of the esophagus or gastrointestinal tract.

The antibody is suitable for use in therapy, for example in treating abreast cancer.

The antibody is suitable for use in therapy, for example in treating afallopian tube cancer.

The antibody is suitable for use in therapy, for example in treating abrain cancer.

The antibody is suitable for use in therapy, for example in treating anurethral cancer.

The antibody is suitable for use in therapy, for example in treating anendometriosis.

The antibody is suitable for use in therapy, for example in treating acervical cancer.

The antibody is suitable for use in therapy, for example in treating ametastatic lesion of the cancer.

The antibody is suitable for use in therapy, for example in treating ahematological malignancy.

The antibody is suitable for use in therapy, for example in treating anon-Hodgkin's lymphoma.

The antibody is suitable for use in therapy, for example in treating achronic lymphocytic leukemia.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with an antagonistic antibody specificallybinding TIM-3.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with an antagonistic antibody specificallybinding TIM-3 comprising the VH of SEQ ID NO: 146 and the VL of SEQ IDNO: 156.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with an antagonistic antibody specificallybinding TIM-3 comprising the VH of SEQ ID NO: 145 and the VL of SEQ IDNO: 155.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with an antagonistic antibody specificallybinding TIM-3 comprising the VH of SEQ ID NO: 172 and the VL of SEQ IDNO: 173.

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with a FGFR inhibitor.

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with a vaccine.

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with an agonistic antibodyspecifically binding GITR (SEQ ID NO: 271).

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with an agonistic antibodyspecifically binding CD137 (SEQ ID NO: 281).

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with an agonistic antibodyspecifically binding OX-40 (SEQ ID NO: 279).

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 66, 67, 68, 69, 70 and 71, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 64 andthe VL of SEQ ID NO: 65.

In some embodiments, the VH and the VL are encoded by polynucleotidesequences of SEQ ID NOs: 198 and 199, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 63 andthe VL of SEQ ID NO: 65.

In some embodiments, the antibody or the antigen-binding portion thereofbinds human PD-1 with an equilibrium dissociation constant (K_(D)) ofless than about 100 nM, optionally less than about 10 nM, for exampleless than about 1 nM such as less than about 100 pM, wherein the K_(D)is measured using ProteOn XPR36 system at +25° C.

In some embodiments, the antibody is an IgG4 isotype, optionallycomprising a S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is an IgG4/K isotype, optionallycomprising the S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 64 andthe VL of SEQ ID NO: 65 and is an IgG4 isotype, optionally comprisingthe S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 64 andthe VL of SEQ ID NO: 65 and is an IgG4K isotype, comprising the S228Psubstitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the HC of SEQ ID NO: 74 andthe LC of SEQ ID NO: 75.

In some embodiments, the antibody is an IgG2 isotype, optionallycomprising V234A, G237A, P238S, H268A, V309L, A330S and P331Ssubstitutions when compared to the wild type IgG2.

In some embodiments, the antibody is an IgG2/K isotype, optionallycomprising V234A, G237A, P238S, H268A, V309L, A330S and P331Ssubstitution when compared to the wild type IgG2.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 64 andthe VL of SEQ ID NO: 65 and is an IgG2/K isotype, optionally comprisingV234A, G237A, P238S, H268A, V309L, A330S and P331S substitution whencompared to the wild type IgG2.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 64 andthe VL of SEQ ID NO: 65 and is an IgG2/K isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitution when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The antibody is suitable for use in therapy, for example in treating amelanoma.

The antibody is suitable for use in therapy, for example in treating alung cancer.

The antibody is suitable for use in therapy, for example in treatingnon-small cell lung cancer (NSCLC).

The antibody is suitable for use in therapy, for example in treating asquamous NSCLC.

The antibody is suitable for use in therapy, for example in treating anon-squamous NSCLC.

The antibody is suitable for use in therapy, for example in treating alung adenocarcinoma.

The antibody is suitable for use in therapy, for example in treating arenal cell carcinoma (RCC).

The antibody is suitable for use in therapy, for example in treating amesothelioma.

The antibody is suitable for use in therapy, for example in treating anasopharyngeal carcinoma (NPC).

The antibody is suitable for use in therapy, for example in treating acolorectal cancer.

The antibody is suitable for use in therapy, for example in treating aprostate cancer.

The antibody is suitable for use in therapy, for example in treating acastration-resistant prostate cancer.

The antibody is suitable for use in therapy, for example in treating astomach cancer.

The antibody is suitable for use in therapy, for example in treating anovarian cancer.

The antibody is suitable for use in therapy, for example in treating agastric cancer.

The antibody is suitable for use in therapy, for example in treating aliver cancer.

The antibody is suitable for use in therapy, for example in treating apancreatic cancer.

The antibody is suitable for use in therapy, for example in treating athyroid cancer.

The antibody is suitable for use in therapy, for example in treating asquamous cell carcinoma of the head and neck.

The antibody is suitable for use in therapy, for example in treating acarcinomas of the esophagus or gastrointestinal tract.

The antibody is suitable for use in therapy, for example in treating abreast cancer.

The antibody is suitable for use in therapy, for example in treating afallopian tube cancer.

The antibody is suitable for use in therapy, for example in treating abrain cancer.

The antibody is suitable for use in therapy, for example in treating anurethral cancer.

The antibody is suitable for use in therapy, for example in treating anendometriosis.

The antibody is suitable for use in therapy, for example in treating acervical cancer.

The antibody is suitable for use in therapy, for example in treating ametastatic lesion of the cancer.

The antibody is suitable for use in therapy, for example in treating ahematological malignancy.

The antibody is suitable for use in therapy, for example in treating anon-Hodgkin's lymphoma.

The antibody is suitable for use in therapy, for example in treating achronic lymphocytic leukemia.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with an antagonistic antibody specificallybinding TIM-3.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with an antagonistic antibody specificallybinding TIM-3 comprising the VH of SEQ ID NO: 146 and the VL of SEQ IDNO: 156.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with an antagonistic antibody specificallybinding TIM-3 comprising the VH of SEQ ID NO: 145 and the VL of SEQ IDNO: 155.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with an antagonistic antibody specificallybinding TIM-3 comprising the VH of SEQ ID NO: 172 and the VL of SEQ IDNO: 173.

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with a FGFR inhibitor.

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with a vaccine.

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with an agonistic antibodyspecifically binding GITR (SEQ ID NO: 271).

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with an agonistic antibodyspecifically binding CD137 (SEQ ID NO: 281).

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with an agonistic antibodyspecifically binding OX-40 (SEQ ID NO: 279).

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-biding portion thereof, comprising the HCDR1,the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:12, 13, 19, 24, 26 and 38, respectively.

In some embodiments, the antibody or the antigen-biding portion thereofcomprises the VH of SEQ ID NO: 47 and the VL of SEQ ID NO: 58.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-biding portion thereof comprising the HCDR1,the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:11, 15, 18, 20, 30 and 32, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 45 and the VL of SEQ ID NO: 60.

In some embodiments, the VH and the VL are encoded by polynucleotidesequences of SEQ ID NOs: 202 and 203, respectively.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the HC of SEQ ID NO: 76 andthe LC of SEQ ID NO: 77.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-biding portion thereof, comprising the HCDR1,the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:10, 13, 16, 20, 26 and 31, respectively.

In some embodiments, the antibody or the antigen-biding portion thereofcomprises the VH of SEQ ID NO: 41 and the VL of SEQ ID NO: 49.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the HC of SEQ ID NO: 212 andthe LC of SEQ ID NO: 213.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-biding portion thereof, comprising the HCDR1,the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:10, 13, 16, 21, 26 and 32, respectively.

In some embodiments, the antibody or the antigen-biding portion thereofcomprises the VH of SEQ ID NO: 41 and the VL of SEQ ID NO: 50.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the HC of SEQ ID NO: 214 andthe LC of SEQ ID NO: 215.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-biding portion thereof, comprising the HCDR1,the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:10, 14, 16, 22, 27 and 33, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 42 and the VL of SEQ ID NO: 51.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the HC of SEQ ID NO: 216 andthe LC of SEQ ID NO: 217.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-biding portion thereof, comprising the HCDR1,the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:10, 14, 16, 22, 26 and 34, respectively.

In some embodiments, the antibody or the antigen-biding portion thereofcomprises the VH of SEQ ID NO: 42 and the VL of SEQ ID NO: 52.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the HC of SEQ ID NO: 218 andthe LC of SEQ ID NO: 219.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 10, 14, 16, 23, 28 and 35, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 42 and the VL of SEQ ID NO: 53.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the HC of SEQ ID NO: 220 andthe LC of SEQ ID NO: 221.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 10, 13, 17, 20, 26 and 31, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 49.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 10, 13, 17, 20, 26 and 36, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 54.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the HC of SEQ ID NO: 222 andthe LC of SEQ ID NO: 223.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 10, 13, 17, 21, 26 and 32, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 50.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the HC of SEQ ID NO: 224 andthe LC of SEQ ID NO: 225.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and theLCDR3 of SEQ ID NOs: 10, 13, 17, 21, 27 and 37, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 55.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the HC of SEQ ID NO: 226 andthe LC of SEQ ID NO: 227.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 10, 13, 17, 23, 26 and 32, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 56.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 10, 13, 17, 22, 26 and 32, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 43 andthe VL of SEQ ID NO: 57.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the HC of SEQ ID NO: 228 andthe LC of SEQ ID NO: 229.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 10, 13, 18, 20, 26 and 31, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 44 and the VL of SEQ ID NO: 49.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 11, 15, 18, 20, 26 and 31, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 45 and the VL of SEQ ID NO: 49.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 10, 13, 19, 20, 26 and 31, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 46 and the VL of SEQ ID NO: 49.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 12, 13, 19, 20, 26 and 31, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 47 and the VL of SEQ ID NO: 49.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 10, 14, 17, 23, 28 and 35, respectively.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 48 andthe VL of SEQ ID NO: 53.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 10, 14, 17, 22, 26 and 34, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 48 and the VL of SEQ ID NO: 52.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 12, 13, 19, 20, 29 and 39, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 47 and the VL of SEQ ID NO: 59.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 11, 15, 18, 25, 26 and 40, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 45 and the VL of SEQ ID NO: 61.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 11, 15, 18, 24, 26 and 32, respectively.

In some embodiments, the antibody of the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 45 and the VL of SEQ ID NO: 62.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype.

In some embodiments, the antibody is an IgG4 isotype comprising theS228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising the VH ofSEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 63 or 64.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising the VL ofSEQ ID NOs: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 or65.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising the VH ofSEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 63 or 64 and the VL of SEQID NOs: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 or 65.

The VH, the VL, the HCDR and the LCDR sequences of exemplaryantagonistic antibodies specifically binding PD-1 of the invention areshown in Table 2.

Although the embodiments illustrated in the Examples comprise pairs ofvariable regions, one from a heavy chain and one from a light chain, askilled artisan will recognize that alternative embodiments may comprisesingle heavy or light chain variable regions. The single variable regionmay be used to screen for variable domains capable of forming atwo-domain specific antigen-binding fragment capable of, for example,binding to human PD-1. The screening may be accomplished by phagedisplay screening methods using for example hierarchical dualcombinatorial approach disclosed in Int. Patent Publ. No. WO1992/01047.In this approach, an individual colony containing either a VH or a VLchain clone is used to infect a complete library of clones encoding theother chain (VL or VH), and the resulting two-chain specificantigen-binding domain is selected in accordance with phage displaytechniques using known methods and those described herein. Therefore,the individual VH and VL polypeptide chains are useful in identifyingadditional antibodies specifically binding to human PD-1 using themethods disclosed in Int. Patent Publ. No. WO1992/01047.

In some embodiments, the antagonistic antibody specifically binding PD-1is a multispecific antibody.

In some embodiments, the antagonistic antibody specifically binding PD-1is a bispecific antibody.

In some embodiments, antagonistic bispecific antibody specificallybinding PD-1 binds PD-L1 (SEQ ID NO: 5), PD-L2 (SEQ ID NO: 8), LAG-3(SEQ ID NO: 293), TIM-3 (SEQ ID NO: 138), CEACAM-1 (SEQ ID NO: 296),CEACAM-5 (SEQ ID NO: 307), OX-40 (SEQ ID NO: 279), GITR (SEQ ID NO:271), CD27 (SEQ ID NO: 280), VISTA (SEQ ID NO: 286), CD137 (SEQ ID NO:281), TIGIT (SEQ ID NO: 301) or CTLA-4 (SEQ ID NO: 292). Bispecific andmultispecific antibodies may be generated using methods describedherein.

TABLE 2 SEQ ID NO: Antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 VH VLPD1B114 10 13 16 20 26 31 41 49 PD1B149 10 13 16 21 26 32 41 50 PD1B16010 14 16 22 27 33 42 51 PD1B162 10 14 16 22 26 34 42 52 PD1B164 10 14 1623 28 35 42 53 PD1B11 10 13 17 20 26 31 43 49 PD1B183 10 13 17 20 26 3643 54 PD1B184 10 13 17 21 26 32 43 50 PD1B185 10 13 17 21 27 37 43 55PD1B187 10 13 17 23 26 32 43 56 PD1B192 10 13 17 22 26 32 43 57 PD1B7110 13 18 20 26 31 44 49 PD1B177 11 15 18 20 26 31 45 49 PD1B70 10 13 1920 26 31 46 49 PD1B175 12 13 19 20 26 31 47 49 PD1B194 10 14 17 23 28 3548 53 PD1B195 10 14 17 22 26 34 48 52 PD1B196 10 14 17 23 26 32 48 56PD1B197 12 13 19 24 26 38 47 58 PD1B198 12 13 19 20 29 39 47 59 PD1B19911 15 18 20 30 32 45 60 PD1B200 11 15 18 25 26 40 45 61 PD1B201 11 15 1824 26 32 45 62 PD1B131 66 67 68 69 70 71 63 65 PD1B132 66 67 68 69 70 7164 65Homologous Antibodies

Variants of the antagonistic antibodies specifically binding PD-1 or theantigen-binding portion thereof of the invention comprising the VH, theVL or the VH and the VL amino acid sequences shown in Table 2, Table 21and Table 22 are within the scope of the invention. For example,variants may comprise one, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acidsubstitutions in the VH and/or the VL as long as the homologousantibodies retain or have improved functional properties when comparedto the parental antibodies. In some embodiments, the sequence identitymay be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to a VHor the VL amino acid sequence of the invention. Optionally, anyvariation of the variant compared to the parental antibody is not withinthe CDRs of the variant.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising the VH ofSEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 63 or 64, the VH optionallyhaving one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions.Optionally, any substitutions are not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising the VL ofSEQ ID NOs: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 or65, the VL optionally having one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen aminoacid substitutions. Optionally, any substitutions are not within theCDRs.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising the VH ofSEQ ID NO: 48 and the VL of SEQ ID NO: 56, wherein the VH, the VL orboth the VH and the VL optionally comprise one, two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen orfifteen amino acid substitutions. Optionally, any substitutions are notwithin the CDRs.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising the VH ofSEQ ID NO: 64 and the VL of SEQ ID NO: 65, wherein the VH, the VL orboth the VH and the VL optionally comprise one, two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen orfifteen amino acid substitutions. Optionally, any substitutions are notwithin the CDRs.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising

the VH of SEQ ID NO: 41 and the VL of SEQ ID NO: 49;

the VH of SEQ ID NO: 41 and the VL of SEQ ID NO: 50;

the VH of SEQ ID NO: 42 and the VL of SEQ ID NO: 51;

the VH of SEQ ID NO: 42 and the VL of SEQ ID NO: 52;

the VH of SEQ ID NO: 42 and the VL of SEQ ID NO: 53;

the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 49;

the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 54;

the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 50;

the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 55;

the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 56;

the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 57;

the VH of SEQ ID NO: 44 and the VL of SEQ ID NO: 49;

the VH of SEQ ID NO: 45 and the VL of SEQ ID NO: 49;

the VH of SEQ ID NO: 46 and the VL of SEQ ID NO: 49;

the VH of SEQ ID NO: 47 and the VL of SEQ ID NO: 49;

the VH of SEQ ID NO: 48 and the VL of SEQ ID NO: 53;

the VH of SEQ ID NO: 48 and the VL of SEQ ID NO: 52;

the VH of SEQ ID NO: 47 and the VL of SEQ ID NO: 58;

the VH of SEQ ID NO: 47 and the VL of SEQ ID NO: 59;

the VH of SEQ ID NO: 45 and the VL of SEQ ID NO: 60;

the VH of SEQ ID NO: 45 and the VL of SEQ ID NO: 61;

the VH of SEQ ID NO: 45 and the VL of SEQ ID NO: 62; or

the VH of SEQ ID NO: 63 and the VL of SEQ ID NO: 65, wherein the VH, theVL or both the VH and the VL optionally comprise one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteenor fifteen amino acid substitutions. Optionally, any substitutions arenot within the CDRs.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising the VHhaving the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the VH of SEQ ID NOs: 41, 42, 43, 44,45, 46, 47, 48, 64 or 65. Optionally, any variation from the sequencesof the SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising the VLhaving the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the VL of SEQ ID NOs: 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62 or 65. Optionally, any variationfrom the sequences of the SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising the VHhaving the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the VH of SEQ ID NOs: 41, 42, 43, 44,45, 46, 47, 48, 63 or 64 and the VL having the amino acid sequence atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical tothe VL of SEQ ID NOs: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62 or 65. Optionally, any variation from the sequences of the SEQ IDNOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising the VHand the VL having the amino acid sequences at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the VH of SEQ ID NO: 48 andthe VL of SEQ ID NO: 56. Optionally, any variation from the sequences ofthe SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising the VHand the VL having the amino acid sequences at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the VH of SEQ ID NO: 64 andthe VL of SEQ ID NO: 65. Optionally, any variation from the sequences ofthe SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising the VHand the VL having the amino acid sequences at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the VH and the VL of SEQ IDNOs:

41 and 49, respectively;

41 and 50, respectively;

42 and 51, respectively;

42 and 52, respectively;

42 and 53, respectively;

43 and 49, respectively;

43 and 54, respectively;

43 and 50, respectively;

43 and 55, respectively;

43 and 56, respectively;

43 and 57, respectively;

44 and 49, respectively;

45 and 49, respectively;

46 and 49, respectively;

47 and 49, respectively;

48 and 53, respectively;

48 and 52, respectively;

47 and 58, respectively;

47 and 59, respectively;

45 and 60, respectively;

45 and 61, respectively;

45 and 62, respectively; or

63 and 65, respectively. Optionally, any variation from the sequences ofthe SEQ ID NOs is not within the CDRs.

The homologous antagonistic antibodies specifically binding PD-1 or theantigen-binding portions thereof of the invention have one, two, three,four or five of the following properties:

-   -   a) enhance an activation of antigen specific CD4⁺ or CD8⁺ T        cells in a dose dependent manner, wherein the activation is        measured using a cytomegalovirus antigen recall assay (CMV        assay) as described in Example 1;    -   b) bind human PD-1 with an equilibrium dissociation constant        (K_(D)) of less than about 100 nM, wherein the K_(D) is measured        using ProteOn XPR36 system at +25° C.;    -   c) bind human PD-1 with the K_(D) of less than about 1 nM,        wherein the K_(D) is measured using ProteOn XPR36 system at +25°        C.;    -   d) bind cynomolgus PD-1 of SEQ ID NO: 3 with the K_(D) of less        than about 100 nM, wherein the K_(D) is measured using ProteOn        XPR36 system at +25° C., or    -   e) bind cynomolgus PD-1 of SEQ ID NO: 3 with the K_(D) of less        than about 1 nM, wherein the K_(D) is measured using ProteOn        XPR36 system at +25° C.

In some embodiments, the antibody enhances activation of antigenspecific CD4⁺ or CD8⁺ T cells in a dose dependent manner, whereinactivation is measured using a cytomegalovirus antigen recall assay (CMVassay) as described in Example 1, and binds human PD-1 with anequilibrium dissociation constant (K_(D)) of less than about 100 nM,wherein the K_(D) is measured using ProteOn XPR36 system at +25° C.

In some embodiments, the antibody enhances activation of antigenspecific CD4⁺ or CD8⁺ T cells in dose dependent manner, whereinactivation is measured using a cytomegalovirus antigen recall assay (CMVassay) as described in Example 1, and binds human PD-1 with anequilibrium dissociation constant (K_(D)) of less than about 10 nM,wherein the K_(D) is measured using ProteOn XPR36 system at +25° C.

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences (i.e., %identity=number of identical positions/total number of positions ×100),taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.

The percent identity between two amino acid sequences may be determinedusing the algorithm of E. Meyers and W. Miller (Comput Appl Biosci4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences may be determined using the Needleman andWunsch (J Mol Biol 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://_www_gcg_com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

Antibodies with Conservative Modifications

The invention also provides antagonistic antibodies specifically bindingPD-1 or antigen-binding portions thereof comprising the VH comprisingthe HCDR1, the HCDR2 and the HCDR3 sequences and the VL comprising theLCDR1, the LCDR2 and the LCDR3 sequences, wherein one or more of the CDRsequences comprise specified amino acid sequences based on theantibodies described herein (e.g., antibodies shown in Table 2, Table 21and Table 22, or conservative modifications thereof, and wherein theantibodies retain the desired functional properties of the parentalantagonistic antibodies specifically binding PD-1 of the invention.

The antibodies with conservative modifications have one, two, three,four or five of the following properties:

-   -   a) enhance an activation of antigen specific CD4⁺ or CD8⁺ T        cells in dose dependent manner, wherein the activation is        measured using a cytomegalovirus antigen recall assay (CMV        assay) as described in Example 1;    -   b) bind human PD-1 with an equilibrium dissociation constant        (K_(D)) of less than about 100 nM, wherein the K_(D) is measured        using ProteOn XPR36 system at +25° C.;    -   c) bind human PD-1 with the K_(D) of less than about 1 nM,        wherein the K_(D) is measured using ProteOn XPR36 system at +25°        C.;    -   d) bind cynomolgus PD-1 of SEQ ID NO: 3 with the K_(D) of less        than about 100 nM, wherein the K_(D) is measured using ProteOn        XPR36 system at +25° C., or e) bind cynomolgus PD-1 of SEQ ID        NO: 3 with the K_(D) of less than about 1 nM, wherein the K_(D)        is measured using ProteOn XPR36 system at +25° C.

In some embodiments, the antibody enhances activation of antigenspecific CD4⁺ or CD8⁺ T cells in dose dependent manner, whereinactivation is measured using a cytomegalovirus antigen recall assay (CMVassay) as described in Example 1, and binds human PD-1 with anequilibrium dissociation constant (K_(D)) of less than about 100 nM,wherein the K_(D) is measured using ProteOn XPR36 system at +25° C.

In some embodiments, the antibody enhances activation of antigenspecific CD4⁺ or CD8⁺ T cells in dose dependent manner, whereinactivation is measured using a cytomegalovirus antigen recall assay (CMVassay) as described in Example 1, and binds human PD-1 with anequilibrium dissociation constant (K_(D)) of less than about 10 nM,wherein the K_(D) is measured using ProteOn XPR36 system at +25° C.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 10, 14, 17, 23, 26 and 32, respectively, and conservativemodifications thereof.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 66, 67, 68, 69, 70 and 71, respectively, and conservativemodifications thereof.

The invention also provides an antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of

SEQ ID NOs: 10, 13, 16, 20, 26 and 31, respectively;

SEQ ID NOs: 10, 13, 16, 21, 26 and 32, respectively;

SEQ ID NOs: 10, 14, 16, 22, 27 and 33, respectively;

SEQ ID NOs: 10, 14, 16, 22, 26 and 34, respectively;

SEQ ID NOs: 10, 14, 16, 23, 28 and 35, respectively;

SEQ ID NOs: 10, 13, 17, 20, 26 and 31, respectively;

SEQ ID NOs: 10, 13, 17, 20, 26 and 36, respectively;

SEQ ID NOs: 10, 13, 17, 21, 26 and 32, respectively;

SEQ ID NOs: 10, 13, 17, 21, 27 and 37, respectively;

SEQ ID NOs: 10, 13, 17, 23, 26 and 32, respectively;

SEQ ID NOs: 10, 13, 17, 22, 26 and 32, respectively;

SEQ ID NOs: 10, 13, 18, 20, 26 and 31, respectively;

SEQ ID NOs: 11, 15, 18, 20, 26 and 31, respectively;

SEQ ID NOs: 10, 13, 19, 20, 26 and 31, respectively;

SEQ ID NOs: 12, 13, 19, 20, 26 and 31, respectively;

SEQ ID NOs: 10, 14, 17, 23, 28 and 35, respectively;

SEQ ID NOs: 10, 14, 17, 22, 26 and 34, respectively;

SEQ ID NOs: 12, 13, 19, 24, 26 and 38, respectively;

SEQ ID NOs: 12, 13, 19, 20, 29 and 39, respectively;

SEQ ID NOs: 11, 15, 18, 20, 30 and 32, respectively;

SEQ ID NOs: 11, 15, 18, 25, 26 and 40, respectively;

SEQ ID NOs: 11, 15, 18, 24, 26 and 32, respectively, and conservativemodifications thereof.

“Conservative modification” refers to amino acid modifications that donot significantly affect or alter the binding characteristics of theantibody containing the amino acid sequences. Conservative modificationsinclude amino acid substitutions, additions and deletions. Conservativesubstitutions are those in which the amino acid is replaced with anamino acid residue having a similar side chain. The families of aminoacid residues having similar side chains are well defined and includeamino acids with acidic side chains (for example, aspartic acid,glutamic acid), basic side chains (for example, lysine, arginine,histidine), nonpolar side chains (for example, alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), uncharged polar sidechains (for example, glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine, tryptophan), aromatic side chains (for example,phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains(for example, glycine, alanine, valine, leucine, isoleucine, serine,threonine), amide (for example, asparagine, glutamine), beta-branchedside chains (for example, threonine, valine, isoleucine) andsulfur-containing side chains (cysteine, methionine). Furthermore, anynative residue in the polypeptide may also be substituted with alanine,as has been previously described for alanine scanning mutagenesis(MacLennan et al., Acta Physiol. Scand. Suppl. 643:55-67, 1998; Sasakiet al., Adv. Biophys. 35:1-24, 1998) Amino acid substitutions to theantibodies of the invention may be made by well-known methods forexample by PCR mutagenesis (U.S. Pat. No. 4,683,195). Alternatively,libraries of variants may be generated using known methods, for exampleusing random (NNK) or non-random codons, for example DVK codons, whichencode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr,Trp). The resulting antibody variants may be tested for theircharacteristics using assays described herein.

Antagonistic Antibodies Specifically Binding TIM-3

T-cell immunoglobulin domain and mucin domain 3 (TIM-3, also known asHepatitis A virus cellular receptor 2 (HAVCR2)) is a co-inhibitoryimmune checkpoint receptor that has been proposed to negatively regulateboth adaptive and innate immune responses. TIM-3 is expressed onspecific subsets of CD4⁺ and CD8⁺ T cells and functions to limit theduration and magnitude of T cell responses.

Multiple lines of evidence support the inhibitory role of TIM-3 inregulating T cell responses. Tim-3-deficient mice exhibit defects in theinduction of both antigen-specific and transplantation tolerance,consistent with TIM-3 inhibiting effector T cells during normal immuneresponses (Sabatos et al., (2003) Nat Immunol 4(11):1102-1110,Sanchez-Fueyo et al., (2003) Nat Immunol 4(11):1093-1101). Anti-TIM-3antibodies exacerbate experimental autoimmune encephalomyelitis (EAE) inanimal models (Monney et al., (2002) Nature 415(6871):536-541). TIM-3has been shown to be a critical driver of the dysfunctional or exhaustedT cell state that occurs in chronic infection and cancer (Sakuishi, K.and A. C. Anderson (2014). Tim-3 Regulation of Cancer Immunity.Tumor-Induced Immune Suppression. D. I. Gabrilovich and A. A. Hurwitz,Springer New York: 239-261).

Blockade of TIM-3 has been shown to restore activity in effector cells,such as cytokine secretion and proliferation. In virally exhausted cellpopulations, e.g., cells infected with HCV, TIM-3-expressing cells(TIM-3⁺ cells) express less TNF-α and IFN-γ cytokines than TIM-3negative cells in both effector cell populations, CD4⁺ and CD8⁺ T cells(Golden-Mason et al., (2009) J Virol 83:9122). Blockade of TIM-3restored proliferation in CD8⁺ T cells from an HIV patient, or in cellsthat recapitulated viral exhaustion (Jones et al., (2008) J Exp Med205:2763), or proliferation and IFN-γ and/or TNF-α secretion in NY-ESO-1specific T cells from PBMCs from metastatic patients (Fourcade et al.,(2010) J Exp Med 207:2175). TIM-3 T cells have been found to beconcentrated in tumors, and contribute to the immunosuppressive tumorenvironment (Sakuishi et al., (2013) Oncoimmunology, 2:e23849).

Blockade of TIM-3 (partially alone and additively or synergistically incombination with PD-1 pathway blockade) has shown anti-tumor efficacy inseveral preclinical cancer models, including CT26 colon carcinoma(Sakuishi et al., (2010) J Exp Med 207(10):2187-94), WT3 sarcoma andTRAMP-C1 prostate carcinoma (Ngiow et al., (2011) Cancer Res71(10):3540-3551).

The mechanisms through which TIM-3 inhibits T cell responses are notfully understood. The cytoplasmic tail of TIM-3 contains multipletyrosine residues (Ferris et al., (2014) J Immunol 193(4): 1525-1530)but lacks inhibitory signaling motifs such as ITIMs or ITSMs that arefound in the PD-1 intracellular tail. The Src family tyrosine kinasesFyn and Lck have been shown to bind to TIM-3, although the exactconsequences of these interactions remain to be confirmed in vivo. Twoopposing models have been proposed for how TIM-3 regulates T cellsignaling. On one hand, TIM-3 has been postulated to negatively regulateTCR signaling by recruiting a phosphatase to the immunological synapse,and de-phosphorylating Lck (Clayton, et al., (2014) J Immunol192(2):782-791). In contrast, TIM-3 has also been proposed to enhanceTCR signaling and paradoxically drive T cells towards a more exhaustedstate, through increased activation of NFAT activity and NFκB signaling.

In addition to expression on effector T cells, TIM-3 is also expressedon regulatory T cells (T-regs) and has been shown to mark a suppressiveT-reg subset in tumors. Analyses using both primary human cells andmouse preclinical models have shown that TIM-3⁺ T-regs are moreeffective at inhibiting T helper1 (Th1) and T helper 17 (Th17) T cellresponses than TIM-3⁻ T-regs (Gautron et al., (2014) Eur J Immunol44(9): 2703-2711; Sakuishi et al., (2013) Oncoimmunology, 2:e23849).Since TIM-3 is expressed on highly suppressive Tregs, it can directlyinhibit CD4⁺ and CD8⁺ T cell responses. In addition, TIM-3⁺ Tregsexpress high levels of IL-10, which has been proposed to driveexhaustion of effector T cells in the TME as an additional indirectmechanism of suppressing anti-tumor immune responses (Sakuishi et al.,(2013) Oncoimmunology, 2:e23849).

TIM-3 is expressed on several innate immune cell types, includingmonocytes/macrophages, dendritic cells, and NK cells. Existing data areconsistent with a suppressive role for TIM-3 in these different celltypes.

TIM-3 is constitutively expressed by circulating CD14⁺ monocytes inhealthy donors, and its expression on peripheral monocytes issignificantly increased in patients with chronic inflammation and cancer(Rong et al., (2014) Tissue Antigens 83(2):76-81). TIM-3 levels are alsoupregulated on macrophages that infiltrate hepatocellular carcinoma(HCC) tumors, compared to macrophages from adjacent tissues, and isproposed to play a role in driving the polarization of macrophages to anM2 tumor-promoting phenotype.

Recently, TIM-3 was reported to be expressed on dendritic cells thatinfiltrate mouse tumors. In this setting, interaction of TIM-3 withHMBG1 was proposed to suppress innate immunity by interfering with therecognition of and response to immunostimulatory nucleic acid (Chiba etal., (2012) Immunol 13(9): 832-842). TIM-3 is also constitutivelyexpressed on NK cells in peripheral blood. A recent study showed that NKcells from advanced melanoma patients express high levels of TIM-3 onperipheral NK cells. Importantly, TIM-3⁺ NK cells were functionallyexhausted and anti-TIM-3 blockade was able to reverse the exhaustion andenhance NK cell functionality (da Silva et al., (2014) Cancer ImmunolRes 2(5): 410-422).

TIM-3 binds ligands galectin-9 (Gal-9), phosphatidylserine (PtdSer),HMGB1 and CEACAM-1. S-type lectin galectin-9 can inhibitTIM-3-associated Th1 effector function and induce apoptosis onTIM-3-expressing T cells in murine models. PtdSer usually resides on theintracellular side of the plasma membrane, but is flipped to theextracellular side during apoptosis. PtdSer binds a preserved cleft inall three human TIM family members (TIM-1, 3, 4) Inhibition of PtdSerbinding to TIM-3 may activate T-cell response. Galectin-9 is secreted bytumor cells and can contribute to evasion from anti-tumor immunity. DNAalarmin HMGB1, for which TIM-3 may act as a “sink,” can prevent theHMGB1/RAGE interactions that stimulate innate immunity. CEACAM-1 caninteract with TIM-3 both in cis as a heterodimer on T cells and in transas a ligand. Interaction between CEACAM-1 and TIM-3 may help mediateblock immune response signaling Co-blockade of TIM-3 and CEACAM-1 inCT26 colon carcinoma showed similar efficacy to that seen forco-blockade of PD-L1 and TIM-3.

Thus, blockade of TIM-3 using the antibodies of the invention describedherein that inhibit TIM-3 function may improve the immune responseagainst infection and anti-tumor immunity.

The invention also provides an isolated antagonistic antibodyspecifically binding TIM-3 or an antigen-binding portion thereof,wherein the antibody inhibits binding of TIM-3 to galectin-9.

Inhibition of binding of TIM-3 to galectin-9 by the antibodies of theinvention may be assessed using competition ELISA. In an exemplaryassay, 1 μg/ml recombinant human Fc-TIM-3 is bound on wells ofmicrotiter plates, the wells are washed and blocked, and 10 μg/ml of thetest antibody is added. Without washing, 7.5 μg/ml galectin-9 is addedinto the wells and incubated for 30 min, after which 0.5 μg/mlanti-galectin-9-biotin antibody is added and incubated for 30 min. Theplates are washed and 0.5 μg/mL neutravidin-HRP conjugate polyclonalantibody is added and incubated for 30 minutes. The plates are washedand POD Chemiluminescence substrate added immediately prior to readingthe luminescence signal. Antibodies of the invention inhibit binding ofTIM-3 to galectin-9 when the binding of galectin-9 is reduced by atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%using an assay described herein and in Example 1. Exemplary antibodiesthat inhibit TIM-3 binding to galectin-9 are antibodies TM3B103,TM3B105, TM3B107, TM3B108, TM3B109, TM3B113, TM3B189, TM3B190 andTM3B196.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 or the antigen-binding portion thereof enhances activation ofantigen specific CD4⁺ or CD8⁺ T cells.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 or the antigen-binding portion thereof enhances an activation ofantigen specific CD4⁺ or CD8⁺ T cells, wherein the activation ofantigen-specific CD4⁺ or CD8⁺ T cells is assessed by measuring astatistically significant enhancement of CD137 surface expression onantigen specific CD4⁺ or CD8⁺ T cells according to methods described inExample 14.

Use of CD137 as a marker of antigen specific CD8⁺ and CD4⁺ T cells thatexpand in response to CMV antigen stimulation allowed the detection ofthe functional effects of the antagonistic TIM-3 antibodies of theinvention.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 or the antigen-binding portion thereof binds TIM-3 within TIM-3residues 32-47

(SEQ ID NO: 261) (WGKGACPVFECGNVVL).

In some embodiments, the antagonistic antibody specifically bindingTIM-3 or the antigen-binding portion thereof binds TIM-3 within TIM-3residues 32-47 (WGKGACPVFECGNVVL) (SEQ ID NO: 261) and residues 50-56(DERDVNY) (SEQ ID NO: 262).

In some embodiments, the antagonistic antibody specifically bindingTIM-3 or the antigen-binding portion thereof binds TIM-3 within TIM-3residues 90-102

(SEQ ID NO: 263) (RIQIPGIMNDEKF).

In some embodiments, the antagonistic antibody specifically bindingTIM-3 or the antigen-binding portion thereof binds TIM-3 within TIM-3residues 90-102 (RIQIPGIMNDEKF) (SEQ ID NO: 263) and residues 50-56(DERDVNY) SEQ ID NO: 262.

“Within” means that 80% or more of the epitope residues the antibodybinds to reside within the recited amino acid stretches, and that up to20% of the epitope residues the antibody binds to reside outside of therecited amino acid stretches.

The Tim-3 epitope the antibody binds to may be resolved for exampleusing hydrogen/deuterium exchange (H/D exchange) or by analyzing acrystal structure of the antibody in complex with TIM-3. The epitoperesidues are those which are protected by the antibody by at least 5%difference in deuteration levels through H/D exchange or those surfaceexposed amino acid residues determined to bind the antibody in a crystalstructure of a complex of the antibody and TIM-3. In the crystalstructure of a complex of the antibody and TIM-3, the epitope residuesare those TIM-3 residues that reside within 4 Å distance or less fromany of the antibody CDR residues.

In an H/D exchange assay, TIM-3 protein is incubated in the presence orabsence of the antibody in deuterated water for predetermined timesresulting in deuterium incorporation at exchangeable hydrogen atomswhich are unprotected by the antibody, followed by protease digestion ofthe protein and analyses of the peptide fragments using LC-MS. In anexemplary assay, 5 μL of the test antibody (10 μg) or 5 μL of thecomplex of TIM-3 and the test antibody (10 and 7.35 μg, respectively) isincubated with 120 μL deuterium oxide labeling buffer (50 mM phosphate,100 mM sodium chloride at pH 7.4) for 0 sec, 60 sec, 300 sec, 1800 sec,7200 sec, and 14400 sec. Deuterium exchange is quenched by adding 63 μLof 5 M guanidine hydrochloride and final pH is 2.5. The quenched sampleis subjected to on-column pepsin/protease type XIII digestion and LC-MSanalysis. For pepsin/protease type XIII digestion, 5 μg of the samplesin 125 μL control buffer (50 mM phosphate, 100 mM sodium chloride at pH7.4) are denatured by adding 63 μL of 5 M guanidine hydrochloride (finalpH is 2.5) and incubating the mixture for 3 min. Then, the mixture issubjected to on-column pepsin/protease type XIII digestion and theresultant peptides analyzed using an UPLC-MS system comprised of aWaters Acquity UPLC coupled to a Q Exactive™ Hybrid Quadrupole-OrbitrapMass Spectrometer (Thermo). Raw MS data is processed using HDXWorkBench, software for the analysis of H/D exchange MS data. Thedeuterium levels are calculated using the average mass differencebetween the deuteriated peptide and its native form (t₀). Peptideidentification is done through searching MS/MS data against the TIM-3sequence with Mascot. The mass tolerance for the precursor and productions is 20 ppm and 0.05 Da, respectively.

For X-ray crystallography, TIM-3 and the test antibody are expressed andpurified using standard protocols. The TIM-3/test antibody complex isincubated overnight at 4° C., concentrated, and separated from theuncomplexed species using size-exclusion chromatography. The complex iscrystallized by the vapor-diffusion method from various known testsolutions for example solutions containing PEG3350, ammonium citrate and2-(N-Morpholino)ethanesulfonic acid (MES).

Antibodies binding within Tim-3 residues 32-47 (WGKGACPVFECGNVVL) (SEQID NO: 261), 90-102 (RIQIPGIMNDEKF) (SEQ ID NO: 263) and/or 50-56(DERDVNY) (SEQ ID NO: 262) may be generated by isolating antibodiesbinding TIM-3 using phage display libraries, selecting those antibodiesthat compete with the reference antibody TM3B105 (VH of SEQ ID NO: 146and VL of SEQ ID NO: 156) or TM3B291 (VH of SEQ ID NO: 172 and VL of SEQID NO: 173) for binding to TIM-3 by 100%, and confirming the epitope ofthe generated antibodies by solving the crystal structure of theantibody/TIM-3 complex. Alternatively, mice, rats or rabbits may beimmunized using peptides encompassing residues 32-47, 90-102 and/or50-56 of TIM-3 and the generated antibodies may be evaluated for theirbinding within the recited region.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof comprising a heavychain complementarity determining region 1 (HCDR1), a HCDR2 and a HCDR3of SEQ ID NOs: 164, 165 and 166, respectively.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof comprising a lightchain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 ofSEQ ID NOs: 167, 168 and 169 respectively.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof comprising theHCDR1, the HCDR2 and the HCDR3 of SEQ ID NOs: 164, 165 and 166,respectively, and the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 167,168 and 169 respectively.

SEQ ID NOs: 164, 165, 166, 167, 168 and 169 represent the HCDR1, theHCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 genus sequences ofTIM-3 antagonists derived from phage display libraries. The genussequences were generated based on structural models that resulted in thesequence alignments given in FIG. 13, FIG. 14, FIG. 15, FIG. 16, FIG. 17and FIG. 18 and summarized herein.

SEQ ID NO: 164 X₁₇YX₁₈MX₁₉,whereinX₁₇ is N, S, G or D;X₁₈ is W or A; andX₁₉ is S or H.

SEQ ID NO: 165 X₂₀IX₂₁X₂₂SGGSX₂₃YYADSVKG,whereinX₂₀ is A or V;X₂₁ is S or K;X₂₂ is G or Y; andX₂₃ is T or K.

SEQ ID NO: 166 X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁DY,whereinX₂₄ is D, S, N, G or E;X₂₅ is H, P, E, T or L;X₂₆ is W, E, N or deleted;X₂₇ is D, P or deleted;X₂₈ is P, Y, D or deleted;X₂₉ is N, A, D, G or deleted;X₃₀ is F, P, R, W or V; andX₃₁ is L or F.

SEQ ID NO: 167 X₃₂X₃₃SQSVX₃₄X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀X₄₁X₄₂LA,whereinX₃₂ is R or K;X₃₃ is A or S;X₃₄ is S, N or L;X₃₅ is S, A, N or deleted;X₃₆ is S or deleted;X₃₇ is S or deleted;X₃₈ is N or deleted;X₃₉ is N or deleted;X₄₀ is K or deleted;X₄₁ is S, D or N; andX₄₂ is Y or T.

SEQ ID NO: 168 X₄₃ASX₄₄RX₄₅X₄₆,whereinX₄₃ is G, D, W or T;X₄₄ is S, N or T;X₄₅ is A or E; andX₄₆ is T or S.

SEQ ID NO: 169 (SEQ ID NO: 169) QQX₄₇X₄₈X₄₉X₅₀PX₅₁T,whereinX₄₇ is Y, G or S;X₄₈ is G or Y;X₄₉ is S, H or T;X₅₀ is S, A or T; andX₅₁ is L, I or W.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof comprising theHCDR1, the HCDR2 and the HCDR3 contained within a heavy chain variableregion (VH) of SEQ ID NOs: 145, 146, 147, 148 or 149, wherein the HCDR1,the HCDR2 and the HCDR3 are defined by Chothia, Kabat, or IMGT.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof comprising theLCDR1, the LCDR2 and the LCDR3 contained within a light chain variableregion (VL) of SEQ ID NOs: 155, 156, 157 or 158, wherein the LCDR1, theLCDR2 and the LCDR3 are defined by Chothia, Kabat, or IMGT.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 or the antigen-binding portion thereof comprises

the HCDR1 of SEQ ID NOs: 90, 91, 92 or 93;

the HCDR2 of SEQ ID NOs: 99, 100 or 101; and

the HCDR3 of SEQ ID NOs: 107, 108, 109, 110 or 111.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 or the antigen-binding portion thereof of the invention comprises

the LCDR1 of SEQ ID NOs: 117, 118, 119 or 120;

the LCDR2 of SEQ ID NOs: 126, 127, 128 or 129; and

the LCDR3 of SEQ ID NOs: 135, 136, 137 or 139.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 or the antigen-binding portion thereof comprises

the HCDR1 of SEQ ID NOs: 90, 91, 92 or 93;

the HCDR2 of SEQ ID NOs: 99, 100 or 101;

the HCDR3 of SEQ ID NOs: 107, 108, 109, 110 or 111;

the LCDR1 of SEQ ID NOs: 117, 118, 119 or 120;

the LCDR2 of SEQ ID NOs: 126, 127, 128 or 129; or

the LCDR3 of SEQ ID NOs: 135, 136, 137 or 139.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 or the antigen-binding portion thereof comprises the HCDR1, theHCDR2 and the HCDR3 of

SEQ ID NOs: 90, 99 and 107, respectively;

SEQ ID NOs: 91, 99 and 108, respectively;

SEQ ID NOs: 91, 99 and 109, respectively;

SEQ ID NOs: 92, 100 and 110, respectively; or

SEQ ID NOs: 93, 101 and 111, respectively;

In some embodiments, the antagonistic antibody specifically bindingTIM-3 or the antigen-binding portion thereof comprises the LCDR1, theLCDR2 and the LCDR3 of

SEQ ID NOs: 117, 126 and 135, respectively;

SEQ ID NOs: 118, 127 and 136, respectively;

SEQ ID NOs: 119, 128 and 137, respectively; or

SEQ ID NOs: 120, 129 and 139, respectively.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 or the antigen-binding portion thereof comprises the HCDR1, theHCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of

SEQ ID NOs: 90, 99, 107, 117, 126 and 135, respectively;

SEQ ID NOs: 91, 99, 108, 118, 127 and 136, respectively;

SEQ ID NOs: 91, 99, 109, 119, 128 and 137, respectively;

SEQ ID NOs: 92, 100, 110, 117, 126 and 135, respectively; or

SEQ ID NOs: 93, 101, 111, 120, 129 and 139, respectively.

The invention also provides an isolated antagonistic antibodyspecifically binding TIM-3 or an antigen-binding portion thereof,comprising the HCDR1, the HCDR2 and, the HCDR3 of SEQ ID NOs: 164, 165and 108, respectively, and the LCDR1, the LCDR2 and the LCDR3 of SEQ IDNOs: 118, 168 and 169 respectively.

The invent ion also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 91, 99, 108, 118, 127 and 136, respectively.

In some embodiments, the antibody specifically binding TIM-3 or theantigen-binding portion thereof binds TIM-3 within TIM-3 residues 32-47

(SEQ ID NO: 261) (WGKGACPVFECGNVVL).

In some embodiments, the antibody specifically binding TIM-3 or theantigen-binding portion thereof binds TIM-3 within TIM-3 residues 32-47(WGKGACPVFECGNVVL) (SEQ ID NO: 261) and residues 50-56 (DERDVNY) SEQ IDNO: 262.

In some embodiments, the antibody specifically binding TIM-3 or theantigen-binding portion thereof inhibits binding of TIM-3 to galectin-9.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises a heavy chain framework derived from IGHV3-23 (SEQ ID NO: 174)and a light chain framework derived from IGKV3-11 (SEQ ID NO: 171).

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 146 and the VL of SEQ ID NO: 156.

In some embodiments, the VH and the VL are encoded by polynucleotidesequences of SEQ ID NOs: 204 and 205, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofenhances activation of antigen specific CD4⁺ or CD8⁺ T cells, whereinactivation of antigen-specific CD4⁺ or CD8⁺ T cells is assessed bymeasuring a statistically significant enhancement of CD137 surfaceexpression on antigen specific CD4⁺ or CD8⁺ T cells according to methodsdescribed in Example 14.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype, optionallycomprising a S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is an IgG4/K isotype, optionallycomprising the S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 146 andthe VL of SEQ ID NO: 156 and is an IgG4 isotype, optionally comprisingthe S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 146 andthe VL of SEQ ID NO: 156 and is an IgG4K isotype comprising the S228Psubstitution when compared to the wild type IgG4.

In some embodiments, the antibody is an IgG2 isotype, optionallycomprising V234A, G237A, P238S, H268A, V309L, A330S and P331Ssubstitutions when compared to the wild type IgG2.

In some embodiments, the antibody is an IgG2/K isotype, optionallycomprising V234A, G237A, P238S, H268A, V309L, A330S and P331Ssubstitutions when compared to the wild type IgG2.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 146 andthe VL of SEQ ID NO: 156 and is an IgG2/K isotype, optionally comprisingV234A, G237A, P238S, H268A, V309L, A330S and P331S substitutions whencompared to the wild type IgG2.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 146 andthe VL of SEQ ID NO: 156 and is an IgG2/K isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody comprises the HC of SEQ ID NO: 78 andthe LC of SEQ ID NO: 79.

In some embodiments, the antibody comprises the HC of SEQ ID NO: 240 andthe LC of SEQ ID NO: 79.

SEQ ID NO: 78 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPYAPLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 79EIVLTQSPATLSLSPGERATLSCRASQSVNDYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQGGHAPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECSEQ ID NO: 240 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPYAPLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The antibody is suitable for use in therapy, for example in treating amelanoma.

The antibody is suitable for use in therapy, for example in treating alung cancer.

The antibody is suitable for use in therapy, for example in treatingnon-small cell lung cancer (NSCLC).

The antibody is suitable for use in therapy, for example in treating asquamous NSCLC.

The antibody is suitable for use in therapy, for example in treating anon-squamous NSCLC.

The antibody is suitable for use in therapy, for example in treating alung adenocarcinoma.

The antibody is suitable for use in therapy, for example in treating arenal cell carcinoma (RCC).

The antibody is suitable for use in therapy, for example in treating amesothelioma.

The antibody is suitable for use in therapy, for example in treating anasopharyngeal carcinoma (NPC).

The antibody is suitable for use in therapy, for example in treating acolorectal cancer.

The antibody is suitable for use in therapy, for example in treating aprostate cancer.

The antibody is suitable for use in therapy, for example in treating acastration-resistant prostate cancer.

The antibody is suitable for use in therapy, for example in treating astomach cancer.

The antibody is suitable for use in therapy, for example in treating anovarian cancer.

The antibody is suitable for use in therapy, for example in treating agastric cancer.

The antibody is suitable for use in therapy, for example in treating aliver cancer. The antibody is suitable for use in therapy, for examplein treating a pancreatic cancer.

The antibody is suitable for use in therapy, for example in treating athyroid cancer.

The antibody is suitable for use in therapy, for example in treating asquamous cell carcinoma of the head and neck.

The antibody is suitable for use in therapy, for example in treating acarcinomas of the esophagus or gastrointestinal tract.

The antibody is suitable for use in therapy, for example in treating abreast cancer.

The antibody is suitable for use in therapy, for example in treating afallopian tube cancer.

The antibody is suitable for use in therapy, for example in treating abrain cancer.

The antibody is suitable for use in therapy, for example in treating anurethral cancer.

The antibody is suitable for use in therapy, for example in treating anendometriosis.

The antibody is suitable for use in therapy, for example in treating acervical cancer.

The antibody is suitable for use in therapy, for example in treating ametastatic lesion of the cancer.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with an antagonistic antibody that specificallybinds PD-1.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with the antagonistic antibody that specificallybinds PD-1 comprising the VH of SEQ ID NO: 48 and the VL of SEQ ID NO:56.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with the antagonistic antibody that specificallybinds PD-1 comprising the VH of SEQ ID NO: 47 and the VL of SEQ ID NO:58.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with the antagonistic antibody that specificallybinds PD-1 comprising the VH of SEQ ID NO: 45 and the VL of SEQ ID NO:60.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with the antagonistic antibody that specificallybinds PD-1 comprising the VH of SEQ ID NO: 64 and the VL of SEQ ID NO:65.

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with an antagonisticantibody specifically binding TIGIT (SEQ ID NO: 301).

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with a FGFR inhibitor.

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with a vaccine.

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with an agonistic antibodyspecifically binding GITR (SEQ ID NO: 271).

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with an agonistic antibodyspecifically binding CD137 (SEQ ID NO: 281).

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with an agonistic antibodyspecifically binding OX-40 (SEQ ID NO: 279).

The antibody is suitable for use in therapy in a subject who is beingtreated or who has been treated with an antagonistic antibodyspecifically binding PD-1 comprising the VH of SEQ ID NO: 230 and the VLof SEQ ID NO: 231. (e.g. KEYTRUDA® (pembrolizumab)).

The antibody is suitable for use in therapy in a subject who is beingtreated or who has been treated with an antagonistic antibodyspecifically binding PD-1 comprising the VH of SEQ ID NO: 232 and the VLof SEQ ID NO: 233. (e.g. OPDIVO® (nivolumab)).

The antibody is suitable for use in therapy in a subject who isrefractory to treatment with the antagonistic antibody specificallybinding PD-1 comprising the VH of SEQ ID NO: 230 and the VL of SEQ IDNO: 231. (e.g. KEYTRUDA® (pembrolizumab)).

The antibody is suitable for use in therapy in a subject who isrefractory to treatment with the antagonistic antibody specificallybinding PD-1 comprising the VH of SEQ ID NO: 232 and the VL of SEQ IDNO: 233. (e.g. OPDIVO® (nivolumab)).

The antibody is suitable for use in therapy in a subject who has arelapsed tumor after treatment with the antagonistic antibodyspecifically binding PD-1 comprising the VH of SEQ ID NO: 230 and the VLof SEQ ID NO: 231. (e.g. KEYTRUDA® (pembrolizumab).

The antibody is suitable for use in therapy in a subject who has arelapsed tumor after treatment with the antagonistic antibodyspecifically binding PD-1 comprising the VH of SEQ ID NO: 232 and the VLof SEQ ID NO: 233. (e.g. OPDIVO® (nivolumab)).

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 97, 105, 115, 124, 133 and 143, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises a heavy chain framework derived from IGHV5-51 (SEQ ID NO: 179)and a light chain framework derived from IGKV1-39 (SEQ ID NO: 182).

In some embodiments, the antibody comprises the VH of SEQ ID NO: 172 andthe VL of SEQ ID NO: 173.

In some embodiments, the VH and the VL are encoded by polynucleotidesequences of SEQ ID NOs: 206 and 207, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofenhances activation of antigen specific CD4⁺ or CD8⁺ T cells, whereinthe activation of antigen-specific CD4⁺ or CD8⁺ T cells is assessed bymeasuring a statistically significant enhancement of CD137 surfaceexpression on antigen specific CD4⁺ or CD8⁺ T cells according to methodsdescribed in Example 14.

In some embodiments, the antibody specifically binding TIM-3 or theantigen-binding portion thereof binds TIM-3 within TIM-3 residues 90-102(RIQIPGIMNDEKF) (SEQ ID NO: 263).

In some embodiments, the antibody specifically binding TIM-3 or theantigen-binding portion thereof binds TIM-3 within TIM-3 residues 90-102(RIQIPGIMNDEKF) (SEQ ID NO: 263) and residues 50-56 (DERDVNY) SEQ ID NO:262.

In some embodiments, the antibody specifically binding TIM-3 or theantigen-binding portion thereof inhibits binding of TIM-3 to galectin-9.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype, optionallycomprising a S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is an IgG4/K isotype, optionallycomprising the S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 172 andthe VL of SEQ ID NO: 173 and is an IgG4 isotype, optionally comprisingthe S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 172 andthe VL of SEQ ID NO: 173 and is an IgG4K isotype comprising the S228Psubstitution when compared to the wild type IgG4.

In some embodiments, the antibody is an IgG2 isotype, optionallycomprising V234A, G237A, P238S, H268A, V309L, A330S and P331Ssubstitutions when compared to the wild type IgG2.

In some embodiments, the antibody is an IgG2/K isotype, optionallycomprising V234A, G237A, P238S, H268A, V309L, A330S and P331Ssubstitutions when compared to the wild type IgG2.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 172 andthe VL of SEQ ID NO: 173 and is an IgG2/K isotype, optionally comprisingV234A, G237A, P238S, H268A, V309L, A330S and P331S substitutions whencompared to the wild type IgG2.

In some embodiments, the antibody comprises the VH of SEQ ID NO: 172 andthe VL of SEQ ID NO: 173 and is an IgG2/K isotype comprising V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2.

In some embodiments, the antibody comprises the HC of SEQ ID NO: 80 andthe LC of SEQ ID NO: 81.

SEQ ID NO: 80 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMQWVRQMPGKGLEWMGAIYPGDGDIRYTQNFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARWEKSTTVVQRNYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ IN NO: 81DIQMTQSPSSLSASVGDRVTITCKASENVGTFVSWYQQKPGKAPKLLIYGASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQSYSYPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC

In some embodiments, the antibody is a bispecific antibody, such as abispecific PD-1/TIM-3 antibody.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The antibody is suitable for use in therapy, for example in treating amelanoma.

The antibody is suitable for use in therapy, for example in treating alung cancer.

The antibody is suitable for use in therapy, for example in treatingnon-small cell lung cancer (NSCLC).

The antibody is suitable for use in therapy, for example in treating asquamous NSCLC.

The antibody is suitable for use in therapy, for example in treating anon-squamous NSCLC.

The antibody is suitable for use in therapy, for example in treating alung adenocarcinoma.

The antibody is suitable for use in therapy, for example in treating arenal cell carcinoma (RCC).

The antibody is suitable for use in therapy, for example in treating amesothelioma.

The antibody is suitable for use in therapy, for example in treating anasopharyngeal carcinoma (NPC).

The antibody is suitable for use in therapy, for example in treating acolorectal cancer.

The antibody is suitable for use in therapy, for example in treating aprostate cancer.

The antibody is suitable for use in therapy, for example in treating acastration-resistant prostate cancer.

The antibody is suitable for use in therapy, for example in treating astomach cancer.

The antibody is suitable for use in therapy, for example in treating anovarian cancer.

The antibody is suitable for use in therapy, for example in treating agastric cancer.

The antibody is suitable for use in therapy, for example in treating aliver cancer.

The antibody is suitable for use in therapy, for example in treating apancreatic cancer.

The antibody is suitable for use in therapy, for example in treating athyroid cancer.

The antibody is suitable for use in therapy, for example in treating asquamous cell carcinoma of the head and neck.

The antibody is suitable for use in therapy, for example in treating acarcinomas of the esophagus or gastrointestinal tract.

The antibody is suitable for use in therapy, for example in treating abreast cancer.

The antibody is suitable for use in therapy, for example in treating afallopian tube cancer.

The antibody is suitable for use in therapy, for example in treating abrain cancer.

The antibody is suitable for use in therapy, for example in treating anurethral cancer.

The antibody is suitable for use in therapy, for example in treating anendometriosis.

The antibody is suitable for use in therapy, for example in treating acervical cancer.

The antibody is suitable for use in therapy, for example in treating ametastatic lesion of the cancer.

The antibody is suitable for use in therapy, for example in treating ahematological malignancy.

The antibody is suitable for use in therapy, for example in treating anacute lymphoblastic leukemia (ALL).

The antibody is suitable for use in therapy, for example in treating acancer, in combination with an antagonistic antibody that specificallybinds PD-1.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with the antagonistic antibody that specificallybinds PD-1 comprising the VH of SEQ ID NO: 48 and the VL of SEQ ID NO:56.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with the antagonistic antibody that specificallybinds PD-1 comprising the VH of SEQ ID NO: 47 and the VL of SEQ ID NO:58.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with the antagonistic antibody that specificallybinds PD-1 comprising the VH of SEQ ID NO: 45 and the VL of SEQ ID NO:60.

The antibody is suitable for use in therapy, for example in treating acancer, in combination with the antagonistic antibody that specificallybinds PD-1 comprising the VH of SEQ ID NO: 65 and the VL of SEQ ID NO:65.

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with an antagonisticantibody specifically binding TIGIT (SEQ ID NO: 301).

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with a FGFR inhibitor.

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with a vaccine.

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with an agonistic antibodyspecifically binding GITR (SEQ ID NO: 271).

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with an agonistic antibodyspecifically binding CD137 (SEQ ID NO: 281).

The antibody is suitable for use in therapy, for example in treatingcancer, such as a solid tumor, in combination with an agonistic antibodyspecifically binding OX-40 (SEQ ID NO: 279).

The antibody is suitable for use in therapy in a subject who is beingtreated or who has been treated with an antagonistic antibodyspecifically binding PD-1 comprising the VH of SEQ ID NO: 230 and the VLof SEQ ID NO: 231. (e.g. KEYTRUDA® (pembrolizumab)).

The antibody is suitable for use in therapy in a subject who is beingtreated or who has been treated with an antagonistic antibodyspecifically binding PD-1 comprising the VH of SEQ ID NO: 232 and the VLof SEQ ID NO: 233. (e.g. OPDIVO® (nivolumab)).

The antibody is suitable for use in therapy in a subject who isrefractory to treatment with the antagonistic antibody specificallybinding PD-1 comprising the VH of SEQ ID NO: 230 and the VL of SEQ IDNO: 231. (e.g. KEYTRUDA® (pembrolizumab)).

The antibody is suitable for use in therapy in a subject who isrefractory to treatment with the antagonistic antibody specificallybinding PD-1 comprising the VH of SEQ ID NO: 232 and the VL of SEQ IDNO: 233. (e.g. OPDIVO® (nivolumab)).

The antibody is suitable for use in therapy in a subject who has arelapsed tumor after treatment with the antagonistic antibodyspecifically binding PD-1 comprising the VH of SEQ ID NO: 230 and the VLof SEQ ID NO: 231. (e.g. KEYTRUDA® (pembrolizumab).

The antibody is suitable for use in therapy in a subject who has arelapsed tumor after treatment with the antagonistic antibodyspecifically binding PD-1 comprising the VH of SEQ ID NO: 232 and the VLof SEQ ID NO: 233. (e.g. OPDIVO® (nivolumab)).

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 90, 99, 107, 117, 126 and 135, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises a heavy chain framework derived from IGHV3-23 (SEQ ID NO: 174)and a light chain framework derived from IGKV3-20 (SEQ ID NO: 180).

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 145 and the VL of SEQ ID NO: 155.

In some embodiments, the VH and the VL are encoded by polynucleotidesequences of SEQ ID NOs: 208 and 209, respectively.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 91, 99, 109, 119, 128 and 137,

In some embodiments, the antibody or the antigen-binding portion thereofcomprises a heavy chain framework derived from IGHV3-23 (SEQ ID NO: 174)and a light chain framework derived from IGKV4-1 (SEQ ID NO: 181).

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 148 and the VL of SEQ ID NO: 157.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 92, 100, 110, 117, 126 and 135, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises a heavy chain framework derived from IGHV3-23 (SEQ ID NO: 174)and a light chain framework derived from IGKV3-20 (SEQ ID NO: 180).

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 147 and the VL of SEQ ID NO: 155.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 93, 101, 111, 120, 129 and 139, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises a heavy chain framework derived from IGHV3-23 (SEQ ID NO: 174)and a light chain framework derived from IGKV3-20 (SEQ ID NO: 180).

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 149 and the VL of SEQ ID NO: 158.

In some embodiments, the VH and the VL are encoded by polynucleotidesequences of SEQ ID NOs: 201 and 211, respectively.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 94, 102, 112, 121, 130 and 140, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises a heavy chain framework derived from IGHV1-02 (SEQ ID NO: 175)and a light chain framework derived from IGKV4-1 (SEQ ID NO: 181).

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 150 and the VL of SEQ ID NO: 159.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 95, 103, 113, 122, 131 and 141, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises a heavy chain framework derived from IGHV4-30-4 (SEQ ID NO:176) and a light chain framework derived from IGKV1-39 (SEQ ID NO: 182).

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 151 and the VL of SEQ ID NO: 160.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 96, 104, 114, 123, 132 and 142, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises a heavy chain framework derived from IGHV1-03 (SEQ ID NO: 177)and a light chain framework derived from IGKV1-33 (SEQ ID NO: 183).

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 152 and the VL of SEQ ID NO: 161.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 97, 105, 115, 124, 133 and 143, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises a heavy chain framework derived from IGHV1-03 (SEQ ID NO: 177)and a light chain framework derived from IGKV1-39 (SEQ ID NO: 182).

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 153 and the VL of SEQ ID NO: 162.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 98, 106, 116, 125, 134 and 144, respectively.

In some embodiments, the antibody or the antigen-binding portion thereofcomprises a heavy chain framework derived from IGHV2-26 (SEQ ID NO: 178)and a light chain framework derived from IGKV4-1 (SEQ ID NO: 181).

In some embodiments, the antibody or the antigen-binding portion thereofcomprises the VH of SEQ ID NO: 154 and the VL of SEQ ID NO: 163.

In some embodiments, the antibody or the antigen-binding portion thereofenhances activation of antigen specific CD4⁺ or CD8⁺ T cells, whereinactivation of antigen-specific CD4⁺ or CD8⁺ T cells is assessed bymeasuring a statistically significant enhancement of CD137 surfaceexpression on antigen specific CD4⁺ or CD8⁺ T cells according to methodsdescribed in Example 14.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype, optionallycomprising a S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is an IgG2 isotype, optionallycomprising V234A, G237A, P238S, H268A, V309L, A330S and P331Ssubstitutions when compared to the wild type IgG2.

The VH, the VL, the HCDR and the LCDR sequences of exemplaryantagonistic antibodies specifically binding TIM-3 of the invention areshown in Table 3.

Although the embodiments illustrated in the Examples comprise pairs ofvariable regions, one from a heavy chain and one from a light chain, askilled artisan will recognize that alternative embodiments may comprisesingle heavy or light chain variable regions. The single variable regionmay be used to screen for variable domains capable of forming atwo-domain specific antigen-binding fragment capable of, for example,binding to human TIM-3. The screening may be accomplished by phagedisplay screening methods similarly as described herein.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 is a multispecific antibody.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 is a bispecific antibody.

In some embodiments, the bispecific or the multispecific antibody bindsPD-1 (SEQ ID NO: 1), PD-L1 (SEQ ID NO: 5), PD-L2 (SEQ ID NO: 8), LAG-3(SEQ ID NO: 293), CEACAM-1 (SEQ ID NO: 296), CEACAM-5 (SEQ ID NO: 307),NKG2D (SEQ ID NO: 282), or TIGIT (SEQ ID NO: 301). Bispecific andmultispecific antibodies may be generated using methods describedherein.

TABLE 3 SEQ ID NO: mAb name HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 VH VLTM3B103 90 99 107 117 126 135 145 155 TM3B105 91 99 108 118 127 136 146156 TM3B109 91 99 109 119 128 137 148 157 TM3B108 92 100 110 117 126 135147 155 TM3B113 93 101 111 120 129 139 149 158 TM3B189 94 102 112 121130 140 150 159 TM3B190 95 103 113 122 131 141 151 160 TM3B193 96 104114 123 132 142 152 161 TM3B195 97 105 115 124 133 143 153 162 TM3B19698 106 116 125 134 144 154 163 TM3B291 97 105 115 124 133 143 172 173Homologous Antibodies

Variants of the antagonistic antibodies specifically binding TIM-3 ofthe invention comprising VH or VL amino acid sequences shown in Table 3,Table 36 and Table 37 are within the scope of the invention. Forexample, variants may comprise one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen aminoacid substitutions in the VH and/or the VL as long as the homologousantibodies retain or have improved functional properties when comparedto the parental antibodies. In some embodiments, the sequence identitymay be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to a VHor the VL amino acid sequence of the invention.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHof SEQ ID NO: 145 and the VL of SEQ ID NO: 155, wherein the VH, the VLor both the VH and the VL optionally comprise one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteenor fifteen amino acid substitutions. Optionally, any substitutions arenot within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHof SEQ ID NO: 146 and the VL of SEQ ID NO: 156, wherein the VH, the VLor both the VH and the VL optionally comprise one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteenor fifteen amino acid substitutions. Optionally, any substitutions arenot within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHof SEQ ID NO: 148 and the VL of SEQ ID NO: 157, wherein the VH, the VLor both the VH and the VL optionally comprise one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteenor fifteen amino acid substitutions. Optionally, any substitutions arenot within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHof SEQ ID NO: 147 and the VL of SEQ ID NO: 155, wherein the VH, the VLor both the VH and the VL optionally comprise one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteenor fifteen amino acid substitutions. Optionally, any substitutions arenot within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHof SEQ ID NO: 149 and the VL of SEQ ID NO: 158, wherein the VH, the VLor both the VH and the VL optionally comprise one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteenor fifteen amino acid substitutions. Optionally, any substitutions arenot within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHof SEQ ID NO: 150 and the VL of SEQ ID NO: 159, wherein the VH, the VLor both the VH and the VL optionally comprise one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteenor fifteen amino acid substitutions. Optionally, any substitutions arenot within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHof SEQ ID NO: 151 and the VL of SEQ ID NO: 160, wherein the VH, the VLor both the VH and the VL optionally comprise one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteenor fifteen amino acid substitutions. Optionally, any substitutions arenot within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHof SEQ ID NO: 152 and the VL of SEQ ID NO: 161, wherein the VH, the VLor both the VH and the VL optionally comprise one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteenor fifteen amino acid substitutions. Optionally, any substitutions arenot within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHof SEQ ID NO: 153 and the VL of SEQ ID NO: 162, wherein the VH, the VLor both the VH and the VL optionally comprise one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteenor fifteen amino acid substitutions. Optionally, any substitutions arenot within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHof SEQ ID NO: 154 and the VL of SEQ ID NO: 163, wherein the VH, the VLor both the VH and the VL optionally comprise one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteenor fifteen amino acid substitutions. Optionally, any substitutions arenot within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHof SEQ ID NO: 172 and the VL of SEQ ID NO: 173, wherein the VH, the VLor both the VH and the VL optionally comprise one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteenor fifteen amino acid substitutions. Optionally, any substitutions arenot within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHhaving the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the VH of SEQ ID NOs: 145, 146, 147,148, 149, 150, 151, 152, 153, 154 or 172. Optionally, any variation fromthe sequences of the SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VLhaving the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the VL of SEQ IS NOs: 155, 156, 157,158, 159, 160, 161, 162, 163 or 173. Optionally, any variation from thesequences of the SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHhaving the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the VH of SEQ ID NOs: 145, 146, 147,148, 149, 150, 151, 152, 153, 154 or 172 and the VL having the aminoacid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the VL of SEQ ID NOs: 155, 156, 157, 158, 159, 160,161, 162, 163 or 173. Optionally, any variation from the sequences ofthe SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHand the VL having the amino acid sequences at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the VH of SEQ ID NO: 145 andthe VL of SEQ ID NO: 155. Optionally, any variation from the sequencesof the SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHand the VL having the amino acid sequences at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the VH of SEQ ID NO: 146 andthe VL of SEQ ID NO: 156. Optionally, any variation from the sequencesof the SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHand the VL having the amino acid sequences at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the VH of SEQ ID NO: 148 andthe VL of SEQ ID NO: 157. Optionally, any variation from the sequencesof the SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHand the VL having the amino acid sequences at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the VH of SEQ ID NO: 147 andthe VL of SEQ ID NO: 155. Optionally, any variation from the sequencesof the SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHand the VL having the amino acid sequences at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the VH of SEQ ID NO: 149 andthe VL of SEQ ID NO: 158. Optionally, any variation from the sequencesof the SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHand the VL having the amino acid sequences at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the VH of SEQ ID NO: 150 andthe VL of SEQ ID NO: 159. Optionally, any variation from the sequencesof the SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHand the VL having the amino acid sequences at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the VH of SEQ ID NO: 151 andthe VL of SEQ ID NO: 160. Optionally, any variation from the sequencesof the SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHand the VL having the amino acid sequences at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the VH of SEQ ID NO: 152 andthe VL of SEQ ID NO: 161. Optionally, any variation from the sequencesof the SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHand the VL having the amino acid sequences at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the VH of SEQ ID NO: 153 andthe VL of SEQ ID NO: 162. Optionally, any variation from the sequencesof the SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHand the VL having the amino acid sequences at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the VH of SEQ ID NO: 154 andthe VL of SEQ ID NO: 163. Optionally, any variation from the sequencesof the SEQ ID NOs is not within the CDRs.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHand the VL having the amino acid sequences at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the VH of SEQ ID NO: 172 andthe VL of SEQ ID NO: 173. Optionally, any variation from the sequencesof the SEQ ID NOs is not within the CDRs.

The homologous antibodies of the invention described herein havesubstantially similar functionality when compared to the parental TIM-3antibodies.

Antagonistic Antibodies Specifically Binding TIM-3 of the Invention withConservative Modifications

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHcomprising the HCDR1, the HCDR2 and the HCDR3 sequences and the VLcomprising the LCDR1, the LCDR2 and the LCDR3 sequences, wherein one ormore of the CDR sequences comprise specified amino acid sequences basedon the antibodies described herein (e.g., antibodies shown in Table 3,Table 36 or Table 37 or conservative modifications thereof, and whereinthe antibodies retain the desired functional properties of the parentalantagonistic antibodies specifically binding TIM-3 of the invention.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 90, 99, 107, 117, 126 and 135, respectively, and conservativemodifications thereof.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 91, 99, 108, 118, 127 and 136, respectively, and conservativemodifications thereof.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 91, 99, 109, 119, 128 and 137, respectively, and conservativemodifications thereof.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 92, 100, 110, 117, 126 and 135, respectively, and conservativemodifications thereof.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 93, 101, 111, 120, 129 and 139, respectively, and conservativemodifications thereof.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 94, 102, 112, 121, 130 and 140, respectively, and conservativemodifications thereof.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 95, 103, 113, 122, 131 and 141, respectively, and conservativemodifications thereof.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 96, 104, 114, 123, 132 and 142, respectively, and conservativemodifications thereof.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 97, 105, 115, 124, 133 and 143, respectively, and conservativemodifications thereof.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 98, 106, 116, 125, 134 and 144, respectively, and conservativemodifications thereof.

“Conservative modification” refers to modifications as described herein.

Antagonistic Antibodies Specifically Binding TIM-3 of the Invention withSpecific Framework Sequences

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHand the VL derived from particular human germline immunoglobulinsequences.

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHframework derived from IGHV3-23 (SEQ ID NO: 174), IGHV1-02 (SEQ ID NO:175), IGHV4-30-4 (SEQ ID NO: 176), IGHV1-03 (SEQ ID NO: 177), IGHV2-26(SEQ ID NO: 178) or IGHV5-51 (SEQ ID NO: 179).

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VLframework derived from IGKV3-20 (A27) (SEQ ID NO: 180), IGKV3-11 (L6)(SEQ ID NO: 171), IGKV4-1 (B3) (SEQ ID NO: 181), IGKV1-39) (012) (SEQ IDNO: 182) or IGKV1-33 (018) (SEQ ID NO: 183).

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHframework derived from IGHV3-23 (SEQ ID NO: 174) and the VL frameworkderived from IGKV3-20 (SEQ ID NO: 180).

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHframework derived from IGHV3-23 (SEQ ID NO: 174) and the VL frameworkderived from IGKV3-11 (SEQ ID NO: 171).

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHframework derived from IGHV3-23 (SEQ ID NO: 174) and the VL frameworkderived from IGKV4-1 (SEQ ID NO: 181).

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHframework derived from IGHV1-02 (SEQ ID NO: 175) and the VL frameworkderived from IGKV4-1 (SEQ ID NO: 181).

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHframework derived from IGHV4-30-4 (SEQ ID NO: 176) and the VL frameworkderived from IGKV1-39 (SEQ ID NO: 182).

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHframework derived from IGHV1-03 (SEQ ID NO: 177) and the VL frameworkderived from IGKV1-33 (SEQ ID NO: 183).

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHframework derived from IGHV1-03 (SEQ ID NO: 177) and the VL frameworkderived from IGKV1-39 (SEQ ID NO: 182).

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHframework derived from IGHV2-26 (SEQ ID NO: 178) and the VL frameworkderived from IGKV4-1 (SEQ ID NO: 181).

The invention also provides an antagonistic antibody specificallybinding TIM-3 or an antigen-binding portion thereof, comprising the VHframework derived from IGHV5-51 (SEQ ID NO: 179) and the VL frameworkderived from IGKV1-39 (SEQ ID NO: 182).

The antibodies of the invention comprising heavy or light chain variableregions “derived from” a particular framework or germline sequence referto antibodies obtained from a system that uses human germlineimmunoglobulin genes, such as from transgenic mice or from phage displaylibraries as discussed herein. An antibody that is “derived from” aparticular framework or germline sequence may contain amino aciddifferences as compared to the sequence it was derived from, due to, forexample, naturally-occurring somatic mutations or intentionalsubstitutions.

Exemplary antagonistic antibodies specifically binding TIM-3 havingcertain VH and VL framework sequences are shown in Table 38.

Bispecific Anti-PD-1/TIM-3 Antibodies

The invention also provides antagonistic bispecific PD-1/TIM-3antibodies.

The invention also provides an isolated antagonistic bispecificPD-1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibody ofthe invention enhances activation of antigen-specific CD4⁺ or CD8⁺ Tcells.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibody ofthe invention enhances activation of antigen-specific CD4⁺ or CD8⁺ Tcells, wherein enhanced activation of antigen-specific CD4⁺ or CD8⁺ Tcells is assessed by measuring a statistically significant increase ofCD137 surface expression on antigen-specific CD4⁺ or CD8⁺ T cells.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibody ofthe invention inhibits TIM-3 binding to galectin-9.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibody ofthe invention

-   -   binds human PD-1 with an equilibrium dissociation constant        (K_(D)) of less than about 100 nM, wherein the K_(D) is measured        using ProteOn XPR36 system at +25° C.;    -   binds human PD-1 with the K_(D) of less than about 1 nM, wherein        the K_(D) is measured using ProteOn XPR36 system at +25° C.;    -   binds cynomolgus PD-1 with the K_(D) of less than about 100 nM,        wherein the K_(D) is measured using ProteOn XPR36 system at +25°        C.; or    -   binds cynomolgus PD-1 with the K_(D) of less than about 1 nM;    -   wherein the K_(D) is measured using ProteOn XPR36 system at +25°        C.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibody ofthe invention enhances an activation of antigen-specific CD4⁺ or CD8⁺ Tcells, wherein the activation of antigen-specific CD4⁺ or CD8⁺ T cellsis assessed by measuring a statistically significant increase of CD137surface expression on antigen-specific CD4⁺ or CD8⁺ T cells and bindshuman PD-1 with an equilibrium dissociation constant (K_(D)) of lessthan about 100 nM, wherein the K_(D) is measured using ProteOn XPR36system at +25° C.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibody ofthe invention enhances the activation of antigen-specific CD4⁺ or CD8⁺ Tcells, wherein the activation of antigen-specific CD4⁺ or CD8⁺ T cellsis assessed by measuring a statistically significant increase of CD137surface expression on antigen-specific CD4⁺ or CD8⁺ T cells, and bindshuman PD-1 with an equilibrium dissociation constant (K_(D)) of lessthan about 1 nM, wherein the K_(D) is measured using ProteOn XPR36system at +25° C.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibody ofthe invention enhances the activation of antigen-specific CD4⁺ or CD8⁺ Tcells, wherein the activation of antigen-specific CD4⁺ or CD8⁺ T cellsis assessed by measuring a statistically significant increase of CD137surface expression on antigen-specific CD4⁺ or CD8⁺ T cells and bindscynomolgus PD-1 with an equilibrium dissociation constant (K_(D)) ofless than about 100 nM, wherein the K_(D) is measured using ProteOnXPR36 system at +25° C.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibody ofthe invention enhances the activation of antigen-specific CD4⁺ or CD8⁺ Tcells, wherein the activation of antigen-specific CD4⁺ or CD8⁺ T cellsis assessed by measuring a statistically significant increase of CD137surface expression on antigen-specific CD4⁺ or CD8⁺ T cells, and bindscynomolgus PD-1 with an equilibrium dissociation constant (K_(D)) ofless than about 1 nM, wherein the K_(D) is measured using ProteOn XPR36system at +25° C.

The antagonistic bispecific PD-1/TIM-3 antibodies of the inventiondescribed herein may be evaluated for their ability to enhance antigenspecific CD4⁺ or CD8⁺ T cell activation, to inhibit TIM-3 binding togalectin-9, and binding kinetics to human or cynomolgus PD-1 or TIM-3may be assessed using methods described herein.

For example, CD137 may be used as a marker for activation of antigenspecific CD4⁺ or CD8⁺ T cells. CD137 surface expression may be measuredon T cells cultured in the presence or in the absence of a testantibody, such as the bispecific PD-1/TIM-3 antibody, using anti-CD137antibody and a secondary antibody conjugated for example to afluorescent dye. The statistically significant difference in theobtained signal on T cells cultured in the presence or in the absence ofthe test antibody is evaluated.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibody ofthe invention binds TIM-3 within TIM-3 residues 32-47 (WGKGACPVFECGNVVL)(SEQ ID NO: 261).

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibody ofthe invention binds TIM-3 within TIM-3 residues 32-47 (WGKGACPVFECGNVVL)(SEQ ID NO: 261) and residues 50-56 (DERDVNY) SEQ ID NO: 262.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibody ofthe invention binds TIM-3 within TIM-3 residues 90-102 (RIQIPGIMNDEKF)(SEQ ID NO: 263).

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibody ofthe invention binds TIM-3 within TIM-3 residues 90-102 (RIQIPGIMNDEKF)(SEQ ID NO: 263) and residues 50-56 (DERDVNY) SEQ ID NO: 262.

In some embodiments, the first domain comprises a heavy chaincomplementarity determining region (HCDR) 1 a HCDR2 and a HCDR3 of SEQID NOs: 82, 83 and 84, respectively.

In some embodiments, the first domain comprises the HCDR1, the HCDR2 andthe HCDR3 of SEQ ID NOs: 82, 83 and 85, respectively.

In some embodiments, the first domain comprises a light chaincomplementarity determining regions (LCDR) 1, a LCDR2 and a LCDR3 of SEQID NOs: 86, 87 and 88, respectively.

In some embodiments, the first domain comprises the HCDR1, the HCDR2 andthe HCDR3 of SEQ ID NOs: 82, 83 and 84, respectively, and the LCDR1, theLCDR2 and the LCDR3 of SEQ ID NOs: 86, 87 and 88, respectively.

In some embodiments, the first domain comprises the HCDR1, the HCDR2 andthe HCDR3 of SEQ ID NOs: 82, 83 and 85, respectively, and the LCDR1, theLCDR2 and the LCDR3 of SEQ ID NOs: 86, 87 and 88, respectively.

In some embodiments, the second domain comprises the HCDR1, the HCDR2and the HCDR3 amino acid sequences of SEQ ID NOs: 164, 165 and 166,respectively.

In some embodiments, the second domain comprises the LCDR1, the LCDR2and the LCDR3 amino acid sequences of SEQ ID NOs: 167, 168 and 169,respectively.

In some embodiments, the second domain comprises the HCDR1, the HCDR2and the HCDR3 amino acid sequences of SEQ ID NOs: 164, 165 and 166,respectively, and the LCDR1, the LCDR2 and the LCDR3 amino acidsequences of SEQ ID NOs: 167, 168 and 169 respectively.

In some embodiments, the first domain comprises the HCDR1, the HCDR2,the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of

SEQ ID NOs: 10, 13, 16, 20, 26 and 31, respectively;

SEQ ID NOs: 10, 13, 16, 21, 26 and 32, respectively;

SEQ ID NOs: 10, 14, 16, 22, 27 and 33, respectively;

SEQ ID NOs: 10, 14, 16, 22, 26 and 34, respectively;

SEQ ID NOs: 10, 14, 16, 23, 28 and 35, respectively;

SEQ ID NOs: 10, 13, 17, 20, 26 and 31, respectively;

SEQ ID NOs: 10, 13, 17, 20, 26 and 36, respectively;

SEQ ID NOs: 10, 13, 17, 21, 26 and 32, respectively;

SEQ ID NOs: 10, 13, 17, 21, 27 and 37, respectively;

SEQ ID NOs: 10, 13, 17, 23, 26 and 32, respectively;

SEQ ID NOs: 10, 13, 17, 22, 26 and 32, respectively;

SEQ ID NOs: 10, 13, 18, 20, 26 and 31, respectively;

SEQ ID NOs: 11, 15, 18, 20, 26 and 31, respectively;

SEQ ID NOs: 10, 13, 19, 20, 26 and 31, respectively;

SEQ ID NOs: 12, 13, 19, 20, 26 and 31, respectively;

SEQ ID NOs: 10, 14, 17, 23, 28 and 35, respectively;

SEQ ID NOs: 10, 14, 17, 22, 26 and 34, respectively;

SEQ ID NOs: 10, 14, 17, 23, 26 and 32, respectively;

SEQ ID NOs: 12, 13, 19, 24, 26 and 38, respectively;

SEQ ID NOs: 12, 13, 19, 20, 29 and 39, respectively;

SEQ ID NOs: 11, 15, 18, 20, 30 and 32, respectively;

SEQ ID NOs: 11, 15, 18, 25, 26 and 40, respectively;

SEQ ID NOs: 11, 15, 18, 24, 26 and 32, respectively; or

SEQ ID NOs: 66, 67, 68, 69, 70 and 71, respectively.

In some embodiments, the second domain comprises the HCDR1, the HCDR2,the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of

SEQ ID NOs: 90, 99, 107, 117, 126 and 135, respectively;

SEQ ID NOs: 91, 99, 108, 118, 127 and 136, respectively;

SEQ ID NOs: 91, 99, 109, 119, 128 and 137, respectively;

SEQ ID NOs: 92, 100, 110, 117, 126 and 135, respectively;

SEQ ID NOs: 93, 101, 111, 120, 129 and 139, respectively;

SEQ ID NOs: 94, 102, 112, 121, 130 and 140, respectively;

SEQ ID NOs: 95, 103, 113, 122, 131 and 141, respectively;

SEQ ID NOs: 96, 104, 114, 123, 132 and 142, respectively;

SEQ ID NOs: 97, 105, 115, 124, 133 and 143, respectively; or

SEQ ID NOs: 98, 106, 116, 125, 134 and 144, respectively.

In some embodiments, the first domain comprises the VH of SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 63 or 64, the VH optionally having one,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen or fifteen conservative amino acid substitutions.Optionally, any substitutions are not within the CDRs.

In some embodiments, the first domain comprises the VL of SEQ ID NOs:49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 or 65, the VLoptionally having one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen or fifteen conservative aminoacid substitutions. Optionally, any substitutions are not within theCDRs.

In some embodiments, the first domain comprises the VH of SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 63 or 64 and the VL of SEQ ID NOs: 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 and 65, the VH, theVL, or the VH and the VL optionally having one, two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen orfifteen conservative amino acid substitutions. Optionally, anysubstitutions are not within the CDRs.

In some embodiments, the second domain comprises the VH of SEQ ID NOs:145, 146, 147, 148, 149, 150, 151, 152, 153, 154 or 172, the VHoptionally having one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen or fifteen conservative aminoacid substitutions. Optionally, any substitutions are not within theCDRs.

In some embodiments, the second domain comprises the VL of SEQ IS NOs:155, 156, 157, 158, 159, 160, 161, 162, 163 or 173, the VL optionallyhaving one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen or fifteen conservative amino acidsubstitutions. Optionally, any substitutions are not within the CDRs.

In some embodiments, the second domain comprises the VH of SEQ ID NOs:145, 146, 147, 148, 149, 150, 151, 152, 153, 154 or 172 and the VL ofSEQ ID NOs: 155, 156, 157, 158, 159, 160, 161, 162, 163 or 173, the VHand the VL optionally having one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteenconservative amino acid substitutions. Optionally, any substitutions arenot within the CDRs.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 41and the VL of SEQ ID NO: 49.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 41and the VL of SEQ ID NO: 50.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 42and the VL of SEQ ID NO: 51.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 42and the VL of SEQ ID NO: 52.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 42and the VL of SEQ ID NO: 53.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 43and the VL of SEQ ID NO: 49.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 43and the VL of SEQ ID NO: 54.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 43and the VL of SEQ ID NO: 50.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 43and the VL of SEQ ID NO: 55.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 43and the VL of SEQ ID NO: 56.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 43and the VL of SEQ ID NO: 57.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 44and the VL of SEQ ID NO: 49.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 45and the VL of SEQ ID NO: 49.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 46and the VL of SEQ ID NO: 49.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 47and the VL of SEQ ID NO: 49.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 48and the VL of SEQ ID NO: 53.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 48and the VL of SEQ ID NO: 52.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 48and the VL of SEQ ID NO: 56.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 47and the VL of SEQ ID NO: 58.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 47and the VL of SEQ ID NO: 59.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 45and the VL of SEQ ID NO: 60.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 45and the VL of SEQ ID NO: 61.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 45and the VL of SEQ ID NO: 62.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 63and the VL of SEQ ID NO: 65.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 64and the VL of SEQ ID NO: 65.

In some embodiments, the second domain comprises the VH of SEQ ID NO:145 and the VL of SEQ ID NO: 155.

In some embodiments, the second domain comprises the VH of SEQ ID NO:146 and the VL of SEQ ID NO: 156.

In some embodiments, the second domain comprises the VH of SEQ ID NO:148 and the VL of SEQ ID NO: 157.

In some embodiments, the second domain comprises the VH of SEQ ID NO:147 and the VL of SEQ ID NO: 155.

In some embodiments, the second domain comprises the VH of SEQ ID NO:149 and the VL of SEQ ID NO: 158.

In some embodiments, the second domain comprises the VH of SEQ ID NO:150 and the VL of SEQ ID NO: 159.

In some embodiments, the second domain comprises the VH of SEQ ID NO:151 and the VL of SEQ ID NO: 160.

In some embodiments, the second domain comprises the VH of SEQ ID NO:152 and the VL of SEQ ID NO: 161.

In some embodiments, the second domain comprises the VH of SEQ ID NO:153 and the VL of SEQ ID NO: 162.

In some embodiments, the second domain comprises the VH of SEQ ID NO:154 and the VL of SEQ ID NO: 163.

In some embodiments, the second domain comprises the VH of SEQ ID NO:172 and the VL of SEQ ID NO: 173.

The invention also provides an isolated antagonistic bispecificPD-1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, wherein the first domaincomprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and theLCDR3 of SEQ ID NOs: 10, 14, 17, 23, 26 and 32, respectively, and thesecond domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 of SEQ ID NOs: 91, 99, 108, 118, 127 and 136,respectively.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibodybinds TIM-3 within TIM-3 residues 32-47 (WGKGACPVFECGNVVL) (SEQ ID NO:261).

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibodybinds TIM-3 within TIM-3 residues 32-47 (WGKGACPVFECGNVVL) (SEQ ID NO:261) and residues 50-56 (DERDVNY) SEQ ID NO: 262.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibodyinhibits TIM-3 binding to galectin-9.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 48and the VL of SEQ ID NO: 56 and the second domain comprises the VH ofSEQ ID NO: 146 and the VL of SEQ ID NO: 156.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising a F405Land/or a K409R substitution.

In some embodiments, the antibody is an IgG2 isotype, optionallycomprising V234A, G237A, P238S, H268A, V309L, A330S and P331Ssubstitutions when compared to the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype, optionallycomprising a S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is an IgG4 isotype comprising a F405Land a K409R substitution.

In some embodiments, the antibody is an IgG4 isotype comprising a heavychain substitution S228P when compared to the wild type IgG4.

In some embodiments, the isolated antagonistic bispecific PD-1/TIM-3antibody comprises a first heavy chain (HC1) a first light chain (LC1),a second heavy chain (HC2) and a second light chain (LC2) of SEQ ID NOs:241, 188, 245 or 194, respectively.

In some embodiments, the isolated antagonistic bispecific PD-1/TIM-3antibody comprises the HC1, the LC1, the HC2 and the LC2 of SEQ ID NOs:186, 188, 191 or 194, respectively.

In some embodiments, the isolated antagonistic bispecific PD-1/TIM-3antibody comprises the HC1, the LC1, the HC2 and the LC2 of SEQ ID NOs:186, 188, 248 or 194, respectively.

In some embodiments, the isolated antagonistic bispecific PD-1/TIM-3antibody comprises the HC1, the LC1, the HC2 and the LC2 of SEQ ID NOs:243, 188, 246 or 194, respectively.

The antibody is suitable for use in therapy, for example in treating acancer.

The antibody is suitable for use in therapy, for example in treating asolid tumor.

The antibody is suitable for use in therapy, for example in treating amelanoma.

The antibody is suitable for use in therapy, for example in treating alung cancer.

The antibody is suitable for use in therapy, for example in treating anon-small cell lung cancer (NSCLC)

The antibody is suitable for use in therapy, for example in treating asquamous NSCLC.

The antibody is suitable for use in therapy, for example in treating anon-squamous NSCLC.

The antibody is suitable for use in therapy, for example in treating alung adenocarcinoma.

The antibody is suitable for use in therapy, for example in treating arenal cell carcinoma (RCC).

The antibody is suitable for use in therapy, for example in treating amesothelioma.

The antibody is suitable for use in therapy, for example in treating anasopharyngeal carcinoma (NPC).

The antibody is suitable for use in therapy, for example in treating acolorectal cancer.

The antibody is suitable for use in therapy, for example in treating aprostate cancer.

The antibody is suitable for use in therapy, for example in treating acastration-resistant prostate cancer.

The antibody is suitable for use in therapy, for example in treating astomach cancer.

The antibody is suitable for use in therapy, for example in treating anovarian cancer.

The antibody is suitable for use in therapy, for example in treating agastric cancer.

The antibody is suitable for use in therapy, for example in treating aliver cancer.

The antibody is suitable for use in therapy, for example in treatingpancreatic cancer.

The antibody is suitable for use in therapy, for example in treating athyroid cancer.

The antibody is suitable for use in therapy, for example in treating asquamous cell carcinoma of the head and neck.

The antibody is suitable for use in therapy, for example in treating acarcinomas of the esophagus or gastrointestinal tract.

The antibody is suitable for use in therapy, for example in treating abreast cancer.

The antibody is suitable for use in therapy, for example in treating afallopian tube cancer.

The antibody is suitable for use in therapy, for example in treating abrain cancer.

The antibody is suitable for use in therapy, for example in treating anurethral cancer.

The antibody is suitable for use in therapy, for example in treating anendometriosis.

The antibody is suitable for use in therapy, for example in treating acervical cancer.

The antibody is suitable for use in therapy, for example in treating ametastatic lesion of the cancer.

The antibody is suitable for use in therapy in a subject who is beingtreated or who has been treated with anti-PD-1 antibody comprising theVH of SEQ ID NO: 230 and the VL of SEQ ID NO: 231. (e.g. KEYTRUDA®(pembrolizumab)).

The antibody is suitable for use in therapy in a subject who is beingtreated or who has been treated with anti-PD-1 antibody comprising theVH of SEQ ID NO: 232 and the VL of SEQ ID NO: 233. (e.g. OPDIVO®(nivolumab)).

The antibody is suitable for use in therapy in a subject who isrefractory to treatment with the anti-PD-1 antibody comprising the VH ofSEQ ID NO: 230 and the VL of SEQ ID NO: 231. (e.g. KEYTRUDA®(pembrolizumab)).

The antibody is suitable for use in therapy in a subject who isrefractory to treatment with the anti-PD-1 antibody comprising the VH ofSEQ ID NO: 232 and the VL of SEQ ID NO: 233. (e.g. OPDIVO® (nivolumab)).

The antibody is suitable for use in therapy in a subject who has arelapsed tumor after treatment with the anti-PD-1 antibody comprisingthe VH of SEQ ID NO: 230 and the VL of SEQ ID NO: 231. (e.g. KEYTRUDA®(pembrolizumab).

The antibody is suitable for use in therapy in a subject who has arelapsed tumor after treatment with the anti-PD-1 antibody comprisingthe VH of SEQ ID NO: 232 and the VL of SEQ ID NO: 233. (e.g. OPDIVO®(nivolumab)).

The invention also provides an isolated antagonistic bispecificPD-1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, wherein the first domaincomprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and theLCDR3 of SEQ ID NOs: 66, 67, 68, 69, 70 and 71, respectively, and thesecond domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 of SEQ ID NOs 97, 105, 115, 124, 133 and 143,respectively.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibodybinds TIM-3 within TIM-3 residues 90-102 (RIQIPGIMNDEKF) (SEQ ID NO:263).

In some embodiments, the bispecific PD-1/TIM-3 antibody binds TIM-3within TIM-3 residues 90-102 (RIQIPGIMNDEKF) (SEQ ID NO: 263) andresidues 50-56 (DERDVNY) SEQ ID NO: 262.

In some embodiments, the bispecific PD-1/TIM-3 antibody inhibits bindingof TIM-3 to galectin-9.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 64and the VL of SEQ ID NO: 65 and the second domain comprises the VH ofSEQ ID NO: 153 and the VL of SEQ ID NO: 162.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising a F405Land/or a K409R substitution.

In some embodiments, the antibody is an IgG2 isotype, optionallycomprising V234A, G237A, P238S, H268A, V309L, A330S and P331Ssubstitutions when compared to the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype, optionallycomprising a S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is an IgG4 isotype comprising a F405Land a K409R substitution.

In some embodiments, the antibody is an IgG4 isotype comprising a heavychain substitution S228P when compared to the wild type IgG4.

In some embodiments, the isolated bispecific PD-1/TIM-3 antibodycomprises the HC1, the LC1, the HC2 and the LC2 of SEQ ID NOs: 187, 189,190 and 193, respectively.

The invention also provides an isolated antagonistic bispecificPD-1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, wherein the first domaincomprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and theLCDR3 of SEQ ID NOs: 66, 67, 68, 69, 70 and 71, respectively, and thesecond domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 of SEQ ID NOs: 91, 99, 108, 118, 127 and 136,respectively.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 64and the VL of SEQ ID NO: 65 and the second domain comprises the VH ofSEQ ID NO: 146 and the VL of SEQ ID NO: 156.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising a F405Land/or a K409R substitution.

In some embodiments, the antibody is an IgG2 isotype, optionallycomprising V234A, G237A, P238S, H268A, V309L, A330S and P331Ssubstitutions when compared to the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype, optionallycomprising a S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is an IgG4 isotype comprising a F405Land a K409R substitution.

In some embodiments, the antibody is an IgG4 isotype comprising a heavychain substitution S228P when compared to the wild type IgG4.

In some embodiments, the isolated antagonistic bispecific PD-1/TIM-3antibody comprises the HC1, the LC1, the HC2 and the LC2 of SEQ ID NOs:187, 189, 191 and 194, respectively.

In some embodiments, the isolated bispecific PD-1/TIM-3 antibodycomprises the HC1, the LC1, the HC2 and the LC2 of SEQ ID NOs: 242, 189,246 and 194, respectively.

The invention also provides an isolated antagonistic bispecificPD-1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, wherein the first domaincomprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and theLCDR3 of SEQ ID NOs: 10, 14, 17, 23, 26 and 32, respectively, and thesecond domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 of SEQ ID NOs: 97, 105, 115, 124, 133 and 143,respectively.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 48and the VL of SEQ ID NO: 56 and the second domain comprises the VH ofSEQ ID NO: 172 and the VL of SEQ ID NO: 173.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising a F405Land/or a K409R substitution.

In some embodiments, the antibody is an IgG2 isotype, optionallycomprising V234A, G237A, P238S, H268A, V309L, A330S and P331Ssubstitutions when compared to the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype, optionallycomprising a S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is an IgG4 isotype comprising a F405Land a K409R substitution.

In some embodiments, the antibody is an IgG4 isotype comprising a heavychain substitution S228P when compared to the wild type IgG4.

In some embodiments, the isolated antagonistic bispecific PD-1/TIM-3antibody comprises the HC1, the LC1, the HC2 and the LC2 of SEQ ID NOs:186, 188, 192 and 195, respectively.

In some embodiments, the isolated antagonistic bispecific PD-1/TIM-3antibody comprises the HC1, the LC1, the HC2 and the LC2 of SEQ ID NOs:241, 188, 244 and 195, respectively.

In some embodiments, the isolated antagonistic bispecific PD-1/TIM-3antibody comprises the HC1, the LC1, the HC2 and the LC2 of SEQ ID NOs:243, 188, 247 and 195, respectively.

In some embodiments, the antibody enhances activation of antigenspecific CD4⁺ or CD8⁺ T cells, wherein activation of antigen-specificCD4⁺ or CD8⁺ T cells is assessed by measuring a statisticallysignificant enhancement of CD137 surface expression on antigen specificCD4⁺ or CD8⁺ T cells according to methods described in Example 14.

The invention also provides an isolated antagonistic bispecificPD-1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3, wherein the first domaincomprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and theLCDR3 of SEQ ID NOs: 10, 14, 17, 23, 26 and 32, respectively, and thesecond domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 of SEQ ID NOs: 97, 105, 115, 124, 133 and 143,respectively.

In some embodiments, the first domain comprises the VH of SEQ ID NO: 48and the VL of SEQ ID NO: 56 and the second domain comprises the VH ofSEQ ID NO: 153 and the VL of SEQ ID NO: 156.

In some embodiments, the antibody is an IgG1 isotype.

In some embodiments, the antibody is an IgG2 isotype.

In some embodiments, the antibody is an IgG2 isotype comprising a F405Land/or a K409R substitution.

In some embodiments, the antibody is an IgG2 isotype, optionallycomprising V234A, G237A, P238S, H268A, V309L, A330S and P331Ssubstitutions when compared to the wild type IgG2.

In some embodiments, the antibody is an IgG3 isotype.

In some embodiments, the antibody is an IgG4 isotype, optionallycomprising a S228P substitution when compared to the wild type IgG4.

In some embodiments, the antibody is an IgG4 isotype comprising a F405Land a K409R substitution.

In some embodiments, the antibody is an IgG4 isotype comprising a heavychain substitution S228P when compared to the wild type IgG4.

In some embodiments, the isolated bispecific PD-1/TIM-3 antibodycomprises the HC1, the LC1, the HC2 and the LC2 of SEQ ID NOs: 186, 188,190 and 193, respectively.

Exemplary antagonistic bispecific PD-1/TIM-3 antibodies of the inventionhaving certain VH, VL, HCDR and LCDR sequences as shown in Table 4 andTable 5.

TABLE 4 PD-1 binding arm SEQ ID NOs: HCDRs LCDRs mAb VH VL 1 2 3 1 2 3PTBB14 48 56 10 14 17 23 26 32 PTBB15 48 56 10 14 17 23 26 32 PTBB16 6465 66 67 68 69 70 71 PTBB17 64 65 66 67 68 69 70 71 PTBB24 48 56 10 1417 23 26 32 PTBB30 48 56 10 14 17 23 26 32 PTBB27 48 56 10 14 17 23 2632 PTBB28 48 56 10 14 17 23 26 32 PTBB18 64 65 66 67 68 69 70 71 PTBB2048 56 10 14 17 23 26 32 PTBB21 48 56 10 14 17 23 26 32

TABLE 5 TIM-3 binding arm SEQ ID NOs: HCDRs LCDR2 mAb VH VL 1 2 3 1 2 3PTBB14 153 162 97 105 115 124 133 143 PTBB15 146 156 91 99 108 118 127136 PTBB16 153 162 97 105 115 124 133 143 PTBB17 146 156 91 99 108 118127 136 PTBB24 172 173 97 105 115 124 133 143 PTBB30 146 156 91 99 108118 127 136 PTBB27 172 173 97 105 115 124 133 143 PTBB28 146 156 91 99108 118 127 136 PTBB18 146 156 91 99 108 118 127 136 PTBB20 146 156 9199 108 118 127 136 PTBB21 172 173 97 105 115 124 133 143Engineered and Modified Antibodies

The antibodies of the invention may further be engineered to generatemodified antibodies with similar or altered properties when compared tothe parental antibodies. The VH, the VL, the VH and the VL, the constantregions, VH framework, VL framework, or any or all of the six CDRs maybe engineered in the antibodies of the invention.

“The antibodies of the invention” as used herein refers to theantagonistic antibodies specifically binding PD-1, the antagonisticantibodies specifically binding TIM-3, and the antagonistic bispecificPD-1/TIM-3 antibodies comprising a first domain specifically bindingPD-1 and a second domain specifically binding TIM-3 (e.g. bispecificPD-1/TIM-3 antibodies) as described herein.

The antibodies of the invention may be engineered by CDR grafting. Oneor more CDR sequences of the antibodies of the invention describedherein may be grafted to a different framework sequence. CDR graftingmay be done using known methods and methods described herein.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 or the bispecific PD-1/TIM-3 antibodies of the invention comprisethe VH that comprises the HDCR1 of SEQ ID NOs: 10, 11 or 12, the HCDR2of SEQ ID NOs: 13, 14 or 15, the HCDR3 of SEQ ID NOs: 16, 17, 18 or 19,and the VL that comprises the LCDR1 of SEQ ID NOs: 20, 21, 22, 23, 24 or25, the LCDR2 of SEQ ID NOs: 26, 27, 28, 29 or 30, and/or the LCDR3 ofSEQ ID NOs: 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, wherein the VHframework is derived from the VH framework other than VH1-69 (SEQ ID NO:170) and the VL framework is derived from the VL framework other thanIGKV3-11 (SEQ ID NO: 171).

In some embodiments, the antagonistic antibodies specifically bindingTIM-3 or the bispecific PD-1/TIM-3 antibodies of the invention comprisethe HDCR1 of SEQ ID NOs: 90, 91, 92, 93, 94, 95, 96, 97 or 98, the HCDR2of SEQ ID NOs: 99, 100, 101, 102, 10, 104, 105 or 106, the HCDR3 of SEQID NOs: 107, 108, 109, 110, 111, 112, 113, 114, 115 or 116, and the VLthat comprises the LCDR1 of SEQ ID NOs: 117, 118, 119, 120, 121, 122,123, 124 or 125, the LCDR2 of SEQ ID NOs: 126, 127, 128, 129, 130, 131,132, 133 or 134, and/or the LCDR3 of SEQ ID NOs: 135, 136, 137, 139,140, 141, 142, 143 or 144, wherein the VH framework is derived from thehuman VH germline gene sequences other than those of IGHV3-23 (SEQ IDNO: 174), IGHV1-02 (SEQ ID NO: 175), IGHV4-30-4 (SEQ ID NO: 176),IGHV1-03 (SEQ ID NO: 177), IGHV2-26 (SEQ ID NO: 178) or IGHV5-51 (SEQ IDNO: 179), and the VL framework is derived from the human VL germlinegene sequences other than those of IGKV3-20 (A27) (SEQ ID NO: 180),IGKV3-11 (L6) (SEQ ID NO: 171), IGKV4-1 (B3) (SEQ ID NO: 181), IGKV1-39(012) (SEQ ID NO: 182) or IGKV1-33 (018) (SEQ ID NO: 183).

The framework sequences to be used may be obtained from public DNAdatabases or published references that include germline antibody genesequences. For example, germline DNA and the encoded protein sequencesof human heavy and light chain variable region genes may be found atIMGT®, the international ImMunoGeneTics Information System®(http://_www-imgt_org). Framework sequences that may be used to replacethe existing framework sequences in the antibodies of the invention maybe those that show the highest percent identity to the parentalframeworks over the entire length of the VH or the VL, or over thelength of the FR1, FR2, FR3 and FR4. In addition, suitable frameworksmay further be selected based on the VH and the VL CDR1 and CDR2 lengthsor identical LCDR1, LCDR2, LCDR3, HCDR1 and HCDR2 canonical structure.Suitable frameworks may be selected using known methods, such as humanframework adaptation described in U.S. Pat. No. 8,748,356 orsuperhumanization described in U.S. Pat. No. 7,709,226.

The framework sequences of the parental and engineered antibodies mayfurther be modified, for example by backmutations to restore and/orimprove binding of the generated antibody to the antigen as describedfor example in U.S. Pat. No. 6,180,370. The framework sequences of theparental or engineered antibodies may further be modified by mutatingone or more residues within the framework region, or within one or moreCDR regions, to remove T-cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and described in further detail in U.S. Patent Publ.No. US20070014796.

The CDR residues of the antibodies of the invention may be mutated toimprove affinity of the antibodies to PD-1, TIM-3, or PD-1 and TIM-3.

The CDR residues of the antibodies of the invention may be mutated forexample to minimize risk of post-translational modifications Amino acidresidues of putative motifs for deamination (NS), acid-catalyzedhydrolysis (DP), isomerization (DS), or oxidation (W) may be substitutedwith any of the naturally occurring amino acids to mutagenize themotifs, and the resulting antibodies may be tested for theirfunctionality and stability using methods described herein.

Fc substitutions may be made to the antibodies of the invention tomodulate antibody effector functions and pharmacokinetic properties. Intraditional immune function, the interaction of antibody-antigencomplexes with cells of the immune system results in a wide array ofresponses, ranging from effector functions such as antibody-dependentcytotoxicity, mast cell degranulation, and phagocytosis toimmunomodulatory signals such as regulating lymphocyte proliferation andantibody secretion. All of these interactions are initiated through thebinding of the Fc domain of antibodies or immune complexes tospecialized cell surface receptors on hematopoietic cells. The diversityof cellular responses triggered by antibodies and immune complexesresults from the structural heterogeneity of the three Fc receptors:FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRI (CD64), FcγRIIA(CD32A) and FcγRIII (CD16) are “activating Fcγ receptors” (i e, immunesystem enhancing); FcγRIIB (CD32B) is an inhibiting Fcγ receptor (i.e.,immune system dampening). Binding to the FcRn receptor modulatesantibody half-life.

In some embodiments, the antagonistic antibodies of the inventioncomprise at least one substitution in an Fc region

In some embodiments, the antagonistic antibodies of the inventioncomprise one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen or

Fc positions that may be substituted to modulate antibody half-life arethose described for example in Dall'Acqua et al., (2006) J Biol Chem281:23514-240, Zalevsky et al., (2010) Nat Biotechnol 28:157-159, Hintonet al., (2004) J Biol Chem 279(8):6213-6216, Hinton et al., (2006) JImmunol 176:346-356, Shields et al. (2001) J Biol Chem 276:6591-6607,Petkova et al., (2006). Int Immunol 18:1759-1769, Datta-Mannan et al.,(2007) Drug Metab Dispos, 35:86-94, 2007, Vaccaro et al., (2005) NatBiotechnol 23:1283-1288, Yeung et al., (2010) Cancer Res, 70:3269-3277and Kim et al., (1999) Eur. J Immunol 29: 2819, and include positions250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434 and 435. Exemplarysubstitutions that may be made singularly or in combination aresubstitutions T250Q, M252Y, 1253A, S254T, T256E, P257I, T307A, D376V,E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R.Exemplary singular or combination substitutions that may be made toincrease the half-life of the antibody are substitutions M428L/N434S,M252Y/S254T/T256E, T250Q/M428L, N434A and T307A/E380A/N434A. Exemplarysingular or combination substitutions that may be made to reduce thehalf-life of the antibody are substitutions H435A, P257I/N434H,D376V/N434H, M252Y/S254T/T256E/H433K/N434F, T308P/N434A and H435R.

In some embodiments, the antibodies of the invention comprise at leastone substitution in the antibody Fc at amino acid position 250, 252,253, 254, 256, 257, 307, 376, 380, 428, 434 or 435.

In some embodiments, the antibodies of the invention comprise at leastone substitution in the antibody Fc selected from the group consistingof T250Q, M252Y, 1253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L,H433K, N434S, N434A, N434H, N434F, H435A and H435R.

In some embodiments, the antibodies of the invention comprise at leastone substitution in the antibody Fc selected from the group consistingof M428L/N434S, M252Y/S254T/T256E, T250Q/M428L, N434A,T307A/E380A/N434A, H435A, P257I/N434H, D376V/N434H,M252Y/S254T/T256E/H433K/N434F, T308P/N434A and H435R.

In some embodiments, the antibodies of the invention comprise at leastone substitution in the antibody Fc that reduces binding of the antibodyto an activating Fcγ receptor (FcγR) and/or reduces Fc effectorfunctions such as C1q binding, complement dependent cytotoxicity (CDC),antibody-dependent cell-mediated cytotoxicity (ADCC) or phagocytosis(ADCP).

Fc positions that may be substituted to reduce binding of the antibodyto the activating FcγR and subsequently to reduce effector function arethose described for example in Shields et al., (2001) J Biol Chem276:6591-6604, Intl. Patent Publ. No. WO2011/066501, U.S. Pat. Nos.6,737,056 and 5,624,821, Xu et al., (2000) Cell Immunol, 200:16-26,Alegre et al., (1994) Transplantation 57:1537-1543, Bolt et al., (1993)Eur J Immunol 23:403-411, Cole et al., (1999) Transplantation,68:563-571, Rother et al., (2007) Nat Biotechnol 25:1256-1264, Ghevaertet al., (2008) J Clin Invest 118:2929-2938, An et al., (2009) mAbs,1:572-579) and include positions 214, 233, 234, 235, 236, 237, 238, 265,267, 268, 270, 295, 297, 309, 327, 328, 329, 330, 331 and 365. Exemplarysubstitutions that may be made singularly or in combination aresubstitutions K214T, E233P, L234V, L234A, deletion of G236, V234A,F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A,N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S and P331Sin IgG1, IgG2, IgG3 or IgG4. Exemplary combination substitutions thatresult in antibodies with reduced ADCC are substitutions L234A/L235A onIgG1, V234A, /G237A/P238S/H268A/V309L/A330S/P331S on IgG2, F234A/L235Aon IgG4, S228P/F234A/L235A on IgG4, N297A on all Ig isotypes,V234A/G237A on IgG2,K214T/E233P/L234V/L235A/G236-deleted/A327G/P331A/D365E/L358M on IgG1,H268Q/V309L/A330S/P331S on IgG2, S267E/L328F on IgG1, L234F/L235E/D265Aon IgG1, L234A/L235A/G237A/P238S/H268A/A330S/P331S on IgG1,S228P/F234A/L235A/G237A/P238S on IgG4, andS228P/F234A/L235A/G236-deleted/G237A/P238S on IgG4. Hybrid IgG2/4 Fcdomains may also be used, such as Fc with residues 117-260 from IgG2 andresidues 261-447 from IgG4.

Well-known S228P substitution may be made in IgG4 antibodies to enhanceIgG4 stability.

In some embodiments, the antibodies of the invention comprise asubstitution in at least one residue position 214, 233, 234, 235, 236,237, 238, 265, 267, 268, 270, 295, 297, 309, 327, 328, 329, 330, 331 or365, wherein residue numbering is according to the EU Index.

In some embodiments, the antibodies of the invention comprise at leastone substitution selected from the group consisting of K214T, E233P,L234V, L234A, deletion of G236, V234A, F234A, L235A, G237A, P238A,P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A,Q295A, V309L, A327S, L328F, A330S and P331 S, wherein residue numberingis according to the EU Index.

In some embodiments, the antibodies of the invention comprise asubstitution in at least one residue position 228, 234, 235, 237, 238,268, 330 or 331, wherein residue numbering is according to the EU Index.

In some embodiments, the antibodies of the invention comprise a S228Psubstitution, wherein residue numbering is according to the EU Index.

In some embodiments, the antibodies of the invention comprise a V234Asubstitution, wherein residue numbering is according to the EU Index.

In some embodiments, the antibodies of the invention comprise a F234Asubstitution, wherein residue numbering is according to the EU Index.

In some embodiments, the antibodies of the invention comprise a G237Asubstitution, wherein residue numbering is according to the EU Index.

In some embodiments, the antibodies of the invention comprise a P238Ssubstitution, wherein residue numbering is according to the EU Index.

In some embodiments, the antibodies of the invention comprise a H268Asubstitution, wherein residue numbering is according to the EU Index.

In some embodiments, the antibodies of the invention comprise a Q268Asubstitution, wherein residue numbering is according to the EU Index.

In some embodiments, the antibodies of the invention comprise an A330Ssubstitution, wherein residue numbering is according to the EU Index.

In some embodiments, the antibodies of the invention comprise a P331Ssubstitution, wherein residue numbering is according to the EU Index.

In some embodiments, the antibodies of the invention comprise L234A,L235A, G237A, P238S, H268A, A330S and P331S substitutions, whereinresidue numbering is according to the EU Index.

In some embodiments, the antibodies of the invention comprise V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions, whereinresidue numbering is according to the EU Index.

In some embodiments, the antibodies of the invention comprise F234A,L235A, G237A, P238S and Q268A substitutions, wherein residue numberingis according to the EU Index.

In some embodiments, the antibodies of the invention comprise L234A,L235A or L234A and L235A substitutions, wherein residue numbering isaccording to the EU Index.

In some embodiments, the antibodies of the invention comprise F234A,L235A or F234A and L235A substitutions, wherein residue numbering isaccording to the EU Index.

In some embodiments, the antibodies of the invention comprise S228P,F234A and L235A substitutions, wherein residue numbering is according tothe EU Index.

In some embodiments, the antibodies of the invention comprise at leastone substitution in an antibody Fc that enhances binding of the antibodyto an Fcγ receptor (FcγR) and/or enhances Fc effector functions such asC1q binding, complement dependent cytotoxicity (CDC), antibody-dependentcell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP).

In addition to their immunomodulatory activity, the PD-1 or the TIM-3antibodies of the invention may kill tumor cells expressing PD-1 and/orTIM-3 directly via antibody-mediated effector functions, for example byADCC, ADCP or CDC.

Fc positions that may be substituted to increase binding of the antibodyto the activating Fcγ and/or enhance antibody effector functions arethose described for example in U.S. Pat. No. 6,737,056, U.S. PatentPubl. No. 2015/0259434, Shields et al., (2001) J Biol Chem276:6591-6604, Lazar et al., (2006) Proc Natal Acad Sci, 103:4005-4010,Stavenhagen et al., (2007) Cancer Res 67:8882-8890, Richards et al.,(2008) Mol Cancer Ther 7:2517-2527, Diebolder et al., Science; publishedonline Mar. 13, 2014; doi:10.1126/science.1248943, and include positions236, 239, 243, 256, 290, 292, 298, 300, 305, 312, 326, 330, 332, 333,334, 345, 360, 339, 378, 396 or 430 (residue numbering according to theEU index). Exemplary substitutions that may be made singularly or incombination are G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L,V305L, K326A, A330K, 1332E, E333A, K334A, A339T and P396L. Exemplarycombination substitutions that result in antibodies with increased ADCCor ADCP are substitutions S239D/1332E, S298A/E333A/K334A,F243L/R292P/Y300L, F243L/R292P/Y300L/P396L,F243L/R292P/Y300L/V3051/P396L and G236A/S239D/1332E on IgG1.

Fc positions that may be substituted to enhance CDC of the antibody arethose described for example in Int. Patent Appl. WO2014/108198, Idusogieet al., (2001) J Immunol 166:2571-2575 and Moore et al., (2010) Mabs,2:181-189, and include positions 267, 268, 324, 326, 333, 345 and 430.Exemplary substitutions that may be made singularly or in combinationare substitutions S267E, H268F, S324T, K326A, K326W, E333A, E345K,E345Q, E345R, E345Y, E430S, E430F and E430T. Exemplary combinationsubstitutions that result in antibodies with increased CDC aresubstitutions K326A/E333A, K326W/E333A, H268F/S324T, S267E/H268F,S267E/S324T and S267E/H268F/S324T on IgG1.

“Antibody-dependent cellular cytotoxicity”, “antibody-dependentcell-mediated cytotoxicity” or “ADCC” is a mechanism for inducing celldeath that depends upon the interaction of antibody-coated target cellswith effector cells possessing lytic activity, such as natural killercells, monocytes, macrophages and neutrophils via Fc gamma receptors(FcγR) expressed on effector cells. For example, NK cells expressFcγRIIIa, whereas monocytes express FcγRI, FcγRII and FcγRIIIa. Death ofthe antibody-coated target cell, such as PD-1 or TIM-3 expressing cells,occurs as a result of effector cell activity through the secretion ofmembrane pore-forming proteins and proteases. To assess ADCC activity ofthe antibody of the invention described herein, the antibody may beadded to TIM-3 or PD-1 expressing cells in combination with immuneeffector cells, which may be activated by the antigen antibody complexesresulting in cytolysis of the target cell. Cytolysis may be detected bythe release of label (e.g. radioactive substrates, fluorescent dyes ornatural intracellular proteins) from the lysed cells. Exemplary effectorcells for such assays include peripheral blood mononuclear cells (PBMC)and NK cells. Exemplary target cells include cells expressing TIM-3 orPD-1 either endogenously or recombinantly. In an exemplary assay, targetcells are used with a ratio of 1 target cell to 50 effector cells.Target cells are pre-labeled with BATDA (PerkinElmer) for 20 minutes at37° C., washed twice and resuspended in DMEM, 10% heat-inactivated FBS,2 mM L-glutamine (all from Invitrogen). Target (1×10⁴ cells) andeffector cells (0.5×10⁶ cells) are combined and 100 id of cells areadded to the wells of 96-well U-bottom plates. An additional 100 μl isadded with or without the test antibodies. The plates are centrifuged at200 g for 3 minutes, incubated at 37° C. for 2 hours, and thencentrifuged again at 200 g for 3 minutes. A total of 20 μl ofsupernatant is removed per well and cell lysis is measured by theaddition of 200 μl of the DELPHIA Europium-based reagent (PerkinElmer).Data is normalized to maximal cytotoxicity with 0.67% Triton X-100(Sigma Aldrich) and minimal control determined by spontaneous release ofBATDA from target cells in the absence of any antibody. The antibody ofthe invention may induce ADCC by about 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

“Antibody-dependent cellular phagocytosis” (“ADCP”) refers to amechanism of elimination of antibody-coated target cells byinternalization by phagocytic cells, such as macrophages or dendriticcells. ADCP may be evaluated by using monocyte-derived macrophages aseffector cells and Daudi cells (ATCC® CCL-213™) or B cell leukemia orlymphoma or tumor cells expressing TIM-3 or PD-1 as target cellsengineered to express GFP or other labeled molecule. Effector:targetcell ratio may be for example 4:1. Effector cells may be incubated withtarget cells for 4 hours with or without the antibody of the invention.After incubation, cells may be detached using accutase. Macrophages maybe identified with anti-CD11b and anti-CD14 antibodies coupled to afluorescent label, and percent phagocytosis may be determined based on %GFP fluorescence in the CD11⁺CD14⁺ macrophages using standard methods.The antibody of the invention may induce ADCP by about 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

“Complement-dependent cytotoxicity”, or “CDC”, refers to a mechanism forinducing cell death in which the Fc effector domain of a target-boundantibody binds and activates complement component C1q which in turnactivates the complement cascade leading to target cell death.Activation of complement may also result in deposition of complementcomponents on the target cell surface that facilitate ADCC by bindingcomplement receptors (e.g., CR3) on leukocytes. CDC of TIM-3 or PD-1expressing cells may be measured for example by plating Daudi cells at1×10⁵ cells/well (50 μl/well) in RPMI-B (RPMI supplemented with 1% BSA),adding 50 μl of test antibodies to the wells at final concentrationbetween 0-100 μg/ml, incubating the reaction for 15 min at roomtemperature, adding 11 μl of pooled human serum to the wells, andincubation the reaction for 45 min at 37° C. Percentage (%) lysed cellsmay be detected as % propidium iodide stained cells in FACS assay usingstandard methods. Antibodies of the invention may induce CDC by about20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or 100%.

The ability of antibodies of the invention described herein to induceADCC may be enhanced by engineering their oligosaccharide component.Human IgG1 or IgG3 are N-glycosylated at Asn297 with the majority of theglycans in the well-known biantennary G0, G0F, G1, G1F, G2 or G2F forms.Antibodies produced by non-engineered CHO cells typically have a glycanfucose content of about at least 85%. The removal of the core fucosefrom the biantennary complex-type oligosaccharides attached to the Fcregions enhances the ADCC of antibodies via improved FcγRIIIa bindingwithout altering antigen binding or CDC activity. Such mAbs may beachieved using different methods reported to lead to the successfulexpression of relatively high defucosylated antibodies bearing thebiantennary complex-type of Fc oligosaccharides such as control ofculture osmolality (Konno et al., (2012) Cytotechnology 64:249-65),application of a variant CHO line Lec13 as the host cell line (Shieldset al., (2002) J Biol Chem 277:26733-26740), application of a variantCHO line EB66 as the host cell line (Olivier et al., MAbs; 2(4), 2010;Epub ahead of print; PMID:20562582), application of a rat hybridoma cellline YB2/0 as the host cell line (Shinkawa et al., (2003) J Biol Chem278:3466-3473), introduction of small interfering RNA specificallyagainst the cc 1,6-fucosyltrasferase (FUT8) gene (Mori et al., (2004)Biotechnol Bioeng 88:901-908), or coexpression ofβ-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II ora potent alpha-mannosidase I inhibitor, kifunensine (Ferrara et al.,(2006) J Biol Chem 281:5032-5036, Ferrara et al., (2006) BiotechnolBioeng 93:851-861; Xhou et al., (2008) Biotechnol Bioeng 99:652-65).

In some embodiments, the antibodies of the invention comprise at leastone substitution in the antibody Fc that enhances effector function ofthe antibody.

In some embodiments, the antibodies of the invention comprise at leastone substitution in the antibody Fc at amino acid position 236, 239,243, 256, 267, 268, 290, 292, 298, 300, 305, 312, 324, 326, 330, 332,333, 334, 345, 360, 339, 378, 396 or 430.

In some embodiments, the antibodies of the invention comprise at leastone substitution in the antibody Fc selected from the group consistingof G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305L, K326A,A330K, 1332E, E333A, K334A, A339T, P396L, S267E, H268F, S324T, K326A,K326W, E333A, E345K, E345Q, E345R, E345Y, E430S, E430F and E430T.

In some embodiments, the antibodies of the invention comprise at leastone substitution in the antibody Fc selected from the group consistingof S239D/I332E, S298A/E333A/K334A, F243L/R292P/Y300L,F243L/R292P/Y300L/P396L, F243L/R292P/Y300L/V3051/P396L,G236A/S239D/1332E, K326A/E333A, K326W/E333A, H268F/S324T, S267E/H268F,S267E/S324T and S267E/H268F/S324T.

In some embodiments, the antibodies of the invention have a biantennaryglycan structure with fucose content of about between 0% to about 15%,for example 15%, 14%, 13%, 12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1% or 0%.

In some embodiments, the antibodies of the invention have a biantennaryglycan structure with fucose content of about 50%, 40%, 45%, 40%, 35%,30%, 25%, 20%, 15%, 14%, 13%, 12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1% or 0%.

Substitutions in the Fc and reduced fucose content may enhance the ADCCactivity of the antagonistic antibodies specifically binding TIM-3 orPD-1 of the invention. TIM-3 or PD-1 antibodies with enhanced ADCC, ADCPand/or CDC activity may be useful in the treatment of patients withTIM-3 and/or PD-1 expressing tumors, including heme malignancies.

“Fucose content” means the amount of the fucose monosaccharide withinthe sugar chain at Asn297. The relative amount of fucose is thepercentage of fucose-containing structures related to allglycostructures. These may be characterized and quantified by multiplemethods, for example: 1) using MALDI-TOF of N-glycosidase F treatedsample (e.g. complex, hybrid and oligo- and high-mannose structures) asdescribed in Intl. Patent Publ. No. WO2008/077546; 2) by enzymaticrelease of the Asn297 glycans with subsequent derivatization anddetection/quantitation by HPLC (UPLC) with fluorescence detection and/orHPLC-MS (UPLC-MS); 3) intact protein analysis of the native or reducedmAb, with or without treatment of the Asn297 glycans with Endo S orother enzyme that cleaves between the first and the second GlcNAcmonosaccharides, leaving the fucose attached to the first GlcNAc; 4)digestion of the mAb to constituent peptides by enzymatic digestion(e.g., trypsin or endopeptidase Lys-C), and subsequent separation,detection and quantitation by HPLC-MS (UPLC-MS) or 5) separation of themAb oligosaccharides from the mAb protein by specific enzymaticdeglycosylation with PNGase F at Asn 297. The oligosaccharides releasedmay be labeled with a fluorophore, separated and identified by variouscomplementary techniques which allow fine characterization of the glycanstructures by matrix-assisted laser desorption ionization (MALDI) massspectrometry by comparison of the experimental masses with thetheoretical masses, determination of the degree of sialylation by ionexchange HPLC (GlycoSep C), separation and quantification of theoligosaccharide forms according to hydrophilicity criteria bynormal-phase HPLC (GlycoSep N), and separation and quantification of theoligosaccharides by high performance capillary electrophoresis-laserinduced fluorescence (HPCE-LIF).

“Low fucose” or “low fucose content” refers to antibodies with fucosecontent of about 0%-15%.

“Normal fucose” or ‘normal fucose content” refers to antibodies withfucose content of about over 50%, typically about over 60%, 70%, 80% orover 85%.

The antibodies of the invention may be post-translationally modified byprocesses such as glycosylation, isomerization, deglycosylation ornon-naturally occurring covalent modification such as the addition ofpolyethylene glycol moieties (pegylation) and lipidation. Suchmodifications may occur in vivo or in vitro. For example, the antibodiesof the invention described herein may be conjugated to polyethyleneglycol (PEGylated) to improve their pharmacokinetic profiles.Conjugation may be carried out by techniques known to those skilled inthe art. Conjugation of therapeutic antibodies with PEG has been shownto enhance pharmacodynamics while not interfering with function (Knighet al., (2004) Platelets 15:409-18; Leong et al., (2001) Cytokine16:106-19; Yang et al., (2003) Protein Eng 16:761-70).

Antibodies of the invention may be modified to improve stability,selectivity, cross-reactivity, affinity, immunogenicity or otherdesirable biological or biophysical property are within the scope of theinvention. Stability of an antibody is influenced by a number offactors, including (1) core packing of individual domains that affectstheir intrinsic stability, (2) protein/protein interface interactionsthat have impact upon the HC and LC pairing, (3) burial of polar andcharged residues, (4) H-bonding network for polar and charged residues;and (5) surface charge and polar residue distribution among other intra-and inter-molecular forces (Worn et al., (2001) J Mol Biol305:989-1010). Potential structure destabilizing residues may beidentified based upon the crystal structure of the antibody or bymolecular modeling in certain cases, and the effect of the residues onantibody stability may be tested by generating and evaluating variantsharboring mutations in the identified residues. One of the ways toincrease antibody stability is to raise the thermal transition midpoint(T_(in)) as measured by differential scanning calorimetry (DSC). Ingeneral, the protein T_(in) is correlated with its stability andinversely correlated with its susceptibility to unfolding anddenaturation in solution and the degradation processes that depend onthe tendency of the protein to unfold (Remmele et al., (2000) Biopharm13:36-46). A number of studies have found correlation between theranking of the physical stability of formulations measured as thermalstability by DSC and physical stability measured by other methods (Guptaet al., (2003) AAPS PharmSci 5E8; Zhang et al., (2004) J Pharm Sci93:3076-89; Maa et al., (1996) Int J Pharm 140:155-68; Bedu-Addo et al.,(2004) Pharm Res 21:1353-61; Remmele et al., (1997) Pharm Res 15:200-8).Formulation studies suggest that a Fab T_(in) has implication forlong-term physical stability of a corresponding mAb.

C-terminal lysine (CTL) may be removed from injected antibodies byendogenous circulating carboxypeptidases in the blood stream (Cai etal., (2011) Biotechnol Bioeng 108:404-412). During manufacturing, CTLremoval may be controlled to less than the maximum level by control ofconcentration of extracellular Zn²⁺, EDTA or EDTA-Fe³⁺ as described inU.S. Patent Publ. No. US20140273092. CTL content in antibodies can bemeasured using known methods.

In some embodiments, the antibodies of the invention have a C-terminallysine content of about 10% to about 90%, about 20% to about 80%, about40% to about 70%, about 55% to about 70%, or about 60%.

In some embodiments, the antibodies of the invention have a C-terminallysine content of about 0%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or 100%.

Methods of Generating Homologous Antibodies, Antibodies withConservative Modifications, and Engineered and Modified Antibodies

The antibodies of the invention that have altered amino acid sequenceswhen compared to the parental antibodies may be generated using standardcloning and expression technologies. For example, site-directedmutagenesis or PCR-mediated mutagenesis may be performed to introducethe mutation(s) and the effect on antibody binding or other property ofinterest, may be evaluated using well known methods and the methodsdescribed herein in the Examples.

Antibody Allotypes

The antibody of the invention may be an IgG1, IgG2, IgG3 or IgG4isotype.

In some embodiments, the antibody of the invention is an IgG1 isotype.

In some embodiments, the antibody of the invention is an IgG2 isotype.

In some embodiments, the antibody of the invention is an IgG3 isotype.

In some embodiments, the antibody of the invention is an IgG4 isotype.

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., (2003) N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,(2011) Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody.

Table 6 shows select IgG1, IgG2 and IgG4 allotypes.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention are of G2m(n), G2m(n−), G2m(n)/(n−), nG4m(a),G1m(17) or G1m(17,1) allotype.

In some embodiments, the antagonistic antibodies specifically bindingTIM-3 of the invention are of G2m(n), G2m(n−), G2m(n)/(n−), nG4m(a),G1m(17) or G1m(17,1) allotype.

In some embodiments, the bispecific PD-1/TIM-3 antibodies of theinvention are of G2m(n), G2m(n−), G2m(n)/(n−), nG4m(a), G1m(17) orG1m(17,1) allotype.

TABLE 6 Amino acid residue at position of diversity (residue numbering:EU Index) IgG2 IgG4 IgG1 Allotype 189 282 309 422 214 356 358 431 G2m(n)T M G2m(n−) P V G2m(n)/(n−) T V nG4m(a) L R G1m(17) K E M A G1m(17,1) KD L AAnti-Idiotypic Antibodies

The present invention provides an anti-idiotypic antibody binding to theantibody of the invention.

The invention also provides an anti-idiotypic antibody specificallybinding to the anti-PD-1 antibody of the invention.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 41 and the VL ofSEQ ID NO: 49.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 41 and the VL ofSEQ ID NO: 50.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 42 and the VL ofSEQ ID NO: 51.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 42 and the VL ofSEQ ID NO: 52.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 42 and the VL ofSEQ ID NO: 53.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 43 and the VL ofSEQ ID NO: 49.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 43 and the VL ofSEQ ID NO: 54.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 43 and the VL ofSEQ ID NO: 50.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 43 and the VL ofSEQ ID NO: 55.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 43 and the VL ofSEQ ID NO: 56.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 43 and the VL ofSEQ ID NO: 57.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 44 and the VL ofSEQ ID NO: 49.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 45 and the VL ofSEQ ID NO: 49.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 46 and the VL ofSEQ ID NO: 49.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 47 and the VL ofSEQ ID NO: 49.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 48 and the VL ofSEQ ID NO: 53.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 48 and the VL ofSEQ ID NO: 52.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 48 and the VL ofSEQ ID NO: 56.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 47 and the VL ofSEQ ID NO: 58.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 47 and the VL ofSEQ ID NO: 59.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 45 and the VL ofSEQ ID NO: 60.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 45 and the VL ofSEQ ID NO: 61.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 45 and the VL ofSEQ ID NO: 62.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 63 and the VL ofSEQ ID NO: 65.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 64 and the VL ofSEQ ID NO: 65.

The invention also provides an anti-idiotypic antibody specificallybinding the antagonistic antibody specifically binding TIM-3 of theinvention.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 145 and the VL ofSEQ ID NO: 155.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 146 and the VL ofSEQ ID NO: 156.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 148 and the VL ofSEQ ID NO: 157.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 147 and the VL ofSEQ ID NO: 155.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 149 and the VL ofSEQ ID NO: 158.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 150 and the VL ofSEQ ID NO: 159.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 151 and the VL ofSEQ ID NO: 160.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 152 and the VL ofSEQ ID NO: 161.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 153 and the VL ofSEQ ID NO: 162.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 154 and the VL ofSEQ ID NO: 163.

In some embodiments, the kit comprises the antagonistic antibodyspecifically binding PD-1 comprising the VH of SEQ ID NO: 48 and the VLof SEQ ID NO: 56.

In some embodiments, the kit comprises the antagonistic antibodyspecifically binding PD-1 comprising the VH of SEQ ID NO: 64 and the VLof SEQ ID NO: 65.

The invention also provides an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 172 and the VL ofSEQ ID NO: 173.

In some embodiments, the anti-idiotypic antibody is used for detectingthe level of the therapeutic antibodies (e.g. anti-PD-1, anti-TIM-3 orthe bispecific PD-1/TIM-3 antibodies of the invention described herein)in a sample.

An anti-idiotypic (Id) antibody is an antibody which recognizes theantigenic determinants (e.g. the paratope or CDRs) of the antibody. TheId antibody may be antigen-blocking or non-blocking. Theantigen-blocking Id may be used to detect the free antibody in a sample(e.g. anti-PD-1, anti-TIM-3 or the bispecific PD-1/TIM-3 antibody of theinvention described herein). The non-blocking Id may be used to detectthe total antibody (free, partially bond to antigen, or fully bound toantigen) in a sample. An Id antibody may be prepared by immunizing ananimal with the antibody to which an anti-Id is being prepared.

An anti-Id antibody may also be used as an immunogen to induce an immuneresponse in yet another animal, producing a so-called anti-anti-Idantibody. An anti-anti-Id may be epitopically identical to the originalmAb, which induced the anti-Id. Thus, by using antibodies to theidiotypic determinants of a mAb, it is possible to identify other clonesexpressing antibodies of identical specificity. Anti-Id antibodies maybe varied (thereby producing anti-Id antibody variants) and/orderivatized by any suitable technique, such as those described elsewhereherein with respect to the antibodies specifically binding PD-1 orTIM-3, or the bispecific PD-1/TIM-3 antibodies.

Immunoconjugates

An “immunoconjugate” refers to the antibody of the invention conjugatedto one or more heterologous molecule(s).

In some embodiments, the antibody of the invention is conjugated to oneor more cytotoxic agents or an imaging agent.

Exemplary cytotoxic agents include chemotherapeutic agents or drugs,growth inhibitory agents, toxins (e.g., protein toxins, enzymaticallyactive toxins of bacterial, fungal, plant, or animal origin, orfragments thereof), and radionuclides.

The cytotoxic agent may be one or more drugs, such as to a mayatansinoid(see, e.g., U.S. Pat. No. 5,208,020, 5,416,06), an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see, e.g.,U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298), a dolastatin, acalicheamicin or derivative thereof (see, e.g., U.S. Pat. Nos.5,712,374, 5,714,586, 5,739, 116, 5,767,285, 5,770,701, 5,770,710,5,773,001, and 5,877,296; Hinman et al., (1993) Cancer Res 53:3336-3342;and Lode et al., (1998) Cancer Res 58:2925-2928); an anthracycline suchas daunomycin or doxorubicin (see, e.g., Kratz et al., (2006) CurrentMed. Chem 13:477-523; Jeffrey et al., (2006) Bioorganic & Med ChemLetters 16:358-362; Torgov et al., (2005) Bioconj Chem 16:717-721; Nagyet al., (2000) Proc Natl Acad Sci USA 97:829-834; Dubowchik et al,Bioorg. & Med. Chem. Letters 12: 1529-1532 (2002); King et al., (2002) JMed Chem 45:4336-4343; and U.S. Pat. No. 6,630,579), methotrexate,vindesine, a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel.

The cytotoxic agent may also be an enzymatically active toxin orfragment thereof, such as diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins, dianthins, Phytolacca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes.

The cytotoxic agent or an imaging agent may also be a radionuclide.Exemplary radionuclides include Ac-225, At-211, 1-131, 1-125, Y-90,Re-186, Re-188, Sm-153, Bi-212, P-32, Pb-212 and radioactive isotopes ofLu. When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example Tc-99m or 1-123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as I-123, I-131, In-111, F-19,C-13, N-15 or O-17.

Conjugates of the antibodies of the invention and the heterologousmolecule may be made using a variety of bifunctional protein couplingagents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HQ), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin maybe prepared as described in Vitetta et al., (1987) Science 238: 1098.Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, e.g., WO94/11026.The linker may be a “cleavable linker” facilitating release of acytotoxic drug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., (1992) Cancer Res 52:127-131; U.S. Pat. No. 5,208,020) may be used.

Conjugates of the antibodies of the invention and the heterologousmolecule may be prepared with cross-linker reagents such as BMPS, EMCS,GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH,sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) whichare commercially available (e.g., from Pierce Biotechnology, Inc.,Rockford, Ill., U.S.A).

The invention also provides an immunoconjugate comprising theantagonistic antibody specifically binding PD-1 of the invention linkedto a therapeutic agent or an imaging agent.

The invention also provides an immunoconjugate comprising theantagonistic antibody specifically binding TIM-3 of the invention linkedto a therapeutic agent or an imaging agent.

The invention also provides an immunoconjugate comprising the bispecificPD-1/TIM-3 antibody of the invention linked to a therapeutic agent or animaging agent.

Generation of Monospecific Antibodies of the Invention

In some embodiments, the antibodies of the invention are human

In some embodiments, the antibodies of the invention are humanized

Monospecific antibodies of the invention described herein (e.g.antibodies specifically binding PD-1 or TIM-3) may be generated usingvarious technologies. For example, the hybridoma method of Kohler andMilstein, Nature 256:495, 1975 may be used to generate monoclonalantibodies. In the hybridoma method, a mouse or other host animal, suchas a hamster, rat or monkey, is immunized with human or cyno PD-1 orTIM-3 or fragments of PD-1 or TIM-3, such as the extracellular domain ofPD-1 or TIM-3, followed by fusion of spleen cells from immunized animalswith myeloma cells using standard methods to form hybridoma cells(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Colonies arising from single immortalizedhybridoma cells are screened for production of antibodies with desiredproperties, such as specificity of binding, cross-reactivity or lackthereof, and affinity for the antigen.

Various host animals may be used to produce the antibodies of theinvention. For example, Balb/c mice may be used to generate mouseanti-human PD-1 or TIM-3 antibodies. The antibodies made in Balb/c miceand other non-human animals may be humanized using various technologiesto generate more human-like sequences.

Exemplary humanization techniques including selection of human acceptorframeworks are known and include CDR grafting (U.S. Pat. No. 5,225,539),SDR grafting (U.S. Pat. No. 6,818,749), Resurfacing (Padlan, (1991) MolImmunol 28:489-499), Specificity Determining Residues Resurfacing (U.S.Patent Publ. No. 2010/0261620), human framework adaptation (U.S. Pat.No. 8,748,356) or superhumanization (U.S. Pat. No. 7,709,226). In thesemethods, CDRs of parental antibodies are transferred onto humanframeworks that may be selected based on their overall homology to theparental frameworks, based on similarity in CDR length, or canonicalstructure identity, or a combination thereof.

Humanized antibodies may be further optimized to improve theirselectivity or affinity to a desired antigen by incorporating alteredframework support residues to preserve binding affinity (backmutations)by techniques such as those described in Int. Patent Publ. Nos.WO1090/007861 and WO1992/22653, or by introducing variation at any ofthe CDRs for example to improve affinity of the antibody.

Transgenic animals, such as mice or rats carrying human immunoglobulin(Ig) loci in their genome may be used to generate human antibodiesagainst a target protein, and are described in for example U.S. Pat. No.6,150,584, Int. Patent Publ. No. WO99/45962, Int. Patent Publ. Nos.WO2002/066630, WO2002/43478, WO2002/043478 and WO1990/04036, Lonberg etal (1994) Nature 368:856-9; Green et al (1994) Nature Genet. 7:13-21;Green & Jakobovits (1998) Exp. Med. 188:483-95; Lonberg and Huszar(1995) Int Rev Immunol 13:65-93; Bruggemann et al., (1991) Eur J Immunol21:1323-1326; Fishwild et al., (1996) Nat Biotechnol 14:845-851; Mendezet al., (1997) Nat Genet 15:146-156; Green (1999) J Immunol Methods231:11-23; Yang et al., (1999) Cancer Res 59:1236-1243; Brtiggemann andTaussig (1997) Curr Opin Biotechnol 8:455-458. The endogenousimmunoglobulin loci in such animal may be disrupted or deleted, and atleast one complete or partial human immunoglobulin locus may be insertedinto the genome of the antimal using homologous or non-homologousrecombination, using transchromosomes, or using minigenes. Companiessuch as Regeneron (http://_www_regeneron_com), Harbour Antibodies(http://_www_harbourantibodies_com), Open Monoclonal Technology, Inc.(OMT) (http://_www_omtincnet), KyMab (http://_www_kymab_com), Trianni(http://_www.trianni_com) and Ablexis (http://_www_ablexis_com) may beengaged to provide human antibodies directed against a selected antigenusing technologies as described above.

Human antibodies may be selected from a phage display library, where thephage is engineered to express human immunoglobulins or portions thereofsuch as Fabs, single chain antibodies (scFv), or unpaired or pairedantibody variable regions (Knappik et al., (2000) J Mol Biol 296:57-86;Krebs et al., (2001) J Immunol Meth 254:67-84; Vaughan et al., (1996)Nature Biotechnology 14:309-314; Sheets et al., (1998) PITAS (USA)95:6157-6162; Hoogenboom and Winter (1991) J Mol Biol 227:381; Marks etal., (1991) J Mol Biol 222:581). The antibodies of the invention may beisolated for example from phage display library expressing antibodyheavy and light chain variable regions as fusion proteins withbacteriophage pIX coat protein as described in Shi et al., (2010)J MolBiol 397:385-96, and Int. Patent Publ. No. WO09/085462). The librariesmay be screened for phage binding to human and/or cyno PD-1 or TIM-3 andthe obtained positive clones may be further characterized, the Fabsisolated from the clone lysates, and expressed as full length IgGs. Suchphage display methods for isolating human antibodies are described infor example: U.S. Pat. Nos. 5,223,409, 5,403,484, 5,571,698, 5,427,908,5,580,717, 5,969,108, 6,172,197, 5,885,793; 6,521,404; 6,544,731;6,555,313; 6,582,915 and 6,593,081.

Preparation of immunogenic antigens and monoclonal antibody productionmay be performed using any suitable technique, such as recombinantprotein production. The immunogenic antigens may be administered to ananimal in the form of purified protein, or protein mixtures includingwhole cells or cell or tissue extracts, or the antigen may be formed denovo in the animal's body from nucleic acids encoding said antigen or aportion thereof.

Generation of Bispecific PD-1/TIM-3 Antibodies of the Invention

The bispecific PD-1/TIM-3 antibodies of the invention (e.g. thebispecific antibodies comprising a first domain specifically bindingPD-1 and a second domain specifically binding TIM-3) may be generated bycombining PD-1 binding VH/VL domains with TIM-3 binding VH/VL domainsisolated and characterized herein. Alternatively, the bispecificPD-1/TIM-3 antibodies may be engineered using VH/VL domains frompublicly available monospecific anti-PD-1 and anti-TIM-3 antibodies,and/or by mix-matching the PD-1 or TIM-3 binding VH/VL domainsidentified herein with publicly available PD-1 or TIM-3 binding VH/VLdomains

Exemplary anti-PD-1 antibodies that may be used to engineer bispecificPD-1/TIM-3 molecules are for example those described in U.S. Pat. Nos.5,897,862 and 7,488,802, and in Int. Patent Publ. Nos. WO2004/004771,WO2004/056875, WO2006/121168, WO2008/156712, WO2010/029435,WO2010/036959, WO2011/110604, WO2012/145493, WO2014/194302,WO2014/206107, WO2015/036394, WO2015/035606, WO2015/085847,WO2015/112900 and WO2015/112805. For example, the VH/VL domains ofKEYTRUDA® (pembrolizumab) and OPDIVO® (nivolumab) may be used. ThesePD-1 VH/VL domains may be incorporated into bispecific antibodiescomprising TIM-3 binding VH/VL domains described herein and in Table 3.For example, the VH/VL domains of the TIM-3 antibodies TM3B103, TM3B105,TM3B107, TM3B108, TM3B109, TM3B113, TM3B189, TM3B190 and TM3B196described herein may be used to generate bispecific PD-1/TIM-3antibodies.

Similarly, exemplary anti-TIM-3 antibodies that may be used to engineerbispecific PD-1/TIM-3 molecules are for example those described in Int.Patent Publ. Nos. WO2011/155607, WO2013/006490, and WO2015/117002. TheseTIM-3 VH/VL domains may be incorporated into bispecific antibodiescomprising PD-1 binding VH/VL domains described herein and in Table 2.For example, the VH/VL domains of the PD-1 antibodies PD1B114, PD1B149,PD1B160, PD1B162, PD1B164, PD1B11, PD1B183, PD1B184, PD1B185, PD1B187,PD1B192, PD1B71, PD1B177, PD1B70, PD1B175, PD1B194, PD1B195, PD1B196,PD1B197, PD1B198, PD1B199, PD1B200, PD1B201, PD1B131 and PD1B132described herein may be used to generate bispecific PD-1/TIM-3antibodies.

The generated bispecific PD-1/TIM-3 antibodies may be tested for theirbinding to PD-1 and TIM-3, and for their desired functionalcharacteristics, such as enhancement of activation of antigen specificCD4⁺ and CD4⁺ T cells using methods described herein.

Bispecific antibodies of the invention comprise antibodies having a fulllength antibody structure.

Full length bispecific antibodies may be generated for example using Fabarm exchange (e g, half molecule exchange, exchanging on heavychain-light chain pair) between two monospecific bivalent antibodies byintroducing mutations at the heavy chain CH3 interface in eachhalf-molecule to favor heterodimer formation of two antibodyhalf-molecules having distinct specificity either in vitro in cell-freeenvironment or using co-expression. The Fab arm exchange reaction is theresult of a disulfide-bond isomerization reaction anddissociation-association of CH3 domains. The heavy chain disulfide bondsin the hinge regions of the parental monospecific antibodies arereduced. The resulting free cysteines of one of the parentalmonospecific antibodies form an inter heavy-chain disulfide bond withcysteine residues of a second parental monospecific antibody moleculeand simultaneously CH3 domains of the parental antibodies release andreform by dissociation-association. The CH3 domains of the Fab arms maybe engineered to favor heterodimerization over homodimerization. Theresulting product is a bispecific antibody having two Fab arms r halfmolecules which each bind a distinct epitope. Mutations F405L in oneheavy chain and K409R in the other heavy chain may be used in case ofIgG1 antibodies. For IgG2 antibodies, a wild-type IgG2 and a IgG2antibody with F405L and R409K substitutions may be used. To generatebispecific antibodies, first monospecific bivalent antibody and thesecond monospecific bivalent antibody are engineered to have a F405L ora K409R mutation in the Fc region, the antibodies are incubated togetherunder reducing conditions sufficient to allow the cysteines in the hingeregion to undergo disulfide bond isomerization; thereby generating thebispecific antibody by Fab arm exchange. The incubation conditions mayoptimally be restored to non-reducing. Exemplary reducing agents thatmay be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT),dithioerythritol (DTE), glutathione, tris(2 carboxyethyl)phosphine(TCEP), L-cysteine and beta-mercaptoethanol. For example, incubation forat least 90 min at a temperature of at least 20° C. in the presence ofat least 25 mM 2-MEA or in the presence of at least 0.5 mMdithiothreitol at a pH of from 5-8, for example at pH of 7.0 or at pH of7.4 may be used.

Bispecific antibodies may also be generated using designs such as theKnob-in-Hole (Genentech), CrossMAbs (Roche) and theelectrostatically-matched (Chugai, Amgen, NovoNordisk, Oncomed), theLUZ-Y (Genentech), the Strand Exchange Engineered Domain body(SEEDbody)(EMD Serono), and the Biclonic (Merus).

The “knob-in-hole” strategy (see, e.g., Intl. Publ. No. WO 2006/028936)may be used to generate full length bispecific antibodies of theinvention. Briefly, selected amino acids forming the interface of theCH3 domains in human IgG can be mutated at positions affecting CH3domain interactions to promote heterodimer formation. An amino acid witha small side chain (hole) is introduced into a heavy chain of anantibody specifically binding a first antigen and an amino acid with alarge side chain (knob) is introduced into a heavy chain of an antibodyspecifically binding a second antigen. After co-expression of the twoantibodies, a heterodimer is formed as a result of the preferentialinteraction of the heavy chain with a “hole” with the heavy chain with a“knob”. Exemplary CH3 substitution pairs forming a knob and a hole are(expressed as modified position in the first CH3 domain of the firstheavy chain/modified position in the second CH3 domain of the secondheavy chain) T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T,T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S_L368A_Y407V.

The CrossMAb technology may be used to generate full length bispecificantibodies of the invention. CrossMAbs, in addition to utilizing the“knob-in-hole” strategy to promoter Fab arm exchange, have in one of thehalf arms the CH1 and the CL domains exchanged to ensure correct lightchain pairing of the resulting bispecific antibody (see e.g. U.S. Pat.No. 8,242,247).

Other cross-over strategies may be used to generate full lengthbispecific antibodies of the invention by exchanging variable orconstant, or both domains between the heavy chain and the light chain orwithin the heavy chain in the bispecific antibodies, either in one orboth arms. These exchanges include for example VH-CH1 with VL-CL, VHwith VL, CH3 with CL and CH3 with CH1 as described in Int. Patent Publ.Nos. WO2009/080254, WO2009/080251, WO2009/018386 and WO2009/080252.

Other strategies such as promoting heavy chain heterodimerization usingelectrostatic interactions by substituting positively charged residuesat one CH3 surface and negatively charged residues at a second CH3surface may be used, as described in US Patent Publ. No. U52010/0015133;US Patent Publ. No. US2009/0182127; US Patent Publ. No. US2010/028637 orUS Patent Publ. No. US2011/0123532. In other strategies,heterodimerization may be promoted by following substitutions (expressedas modified positions in the first CH3 domain of the first heavychain/modified position in the second CH3 domain of the second heavychain) L351Y_F405A_Y407V/T394W, T366I_K392M_T394W/F405A_Y407V,T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K409F,L351Y_Y407A/T366V_K409F, Y407A/T366A_K409F, orT350V_L351Y_F405A_Y407V/T350V_T366L_K392L_T394W as described in U.S.Patent Publ. No. US2012/0149876 or U.S. Patent Publ. No. US2013/0195849.

LUZ-Y technology may be utilized to generate bispecific antibodies ofthe invention. In this technology, a leucine zipper is added into the Cterminus of the CH3 domains to drive the heterodimer assembly fromparental mAbs that is removed post-purification as described in Wraniket al., (2012) J Biol Chem 287(52): 42221-9.

SEEDbody technology may be utilized to generate bispecific antibodies ofthe invention. SEEDbodies have, in their constant domains, select IgGresidues substituted with IgA residues to promote heterodimerization asdescribed in U.S. Patent No. US20070287170.

Mutations are typically made at the DNA level to a molecule such as theconstant domain of the antibody using standard methods.

The antibodies of the invention may be engineered into various wellknown antibody formats.

In some embodiments, the bispecific antibodies include recombinantIgG-like dual targeting molecules, wherein the two sides of the moleculeeach contain the Fab fragment or part of the Fab fragment of at leasttwo different antibodies; IgG fusion molecules, wherein full length IgGantibodies are fused to an extra Fab fragment or parts of Fab fragment;Fc fusion molecules, wherein single chain Fv molecules or stabilizeddiabodies are fused to heavy-chain constant-domains, Fc-regions or partsthereof; Fab fusion molecules, wherein different Fab-fragments are fusedtogether; ScFv- and diabody-based and heavy chain antibodies (e.g.,domain antibodies, nanobodies) wherein different single chain Fvmolecules or different diabodies or different heavy-chain antibodies(e.g. domain antibodies, nanobodies) are fused to each other or toanother protein or carrier molecule.

Polynucleotides, Vectors and Host Cells

The invention also provides an antagonistic antibody that specificallybinds PD-1, TIM-3 or PD-1 and TIM-3 having certain VH and VL sequences,wherein the antibody VH is encoded by a first polynucleotide and theantibody VL is encoded by a second polynucleotide. The polynucleotidemay be a complementary deoxynucleic acid (cDNA), and may be codonoptimized for expression in suitable host. Codon optimization is awell-known technology.

The invention also provides an isolated polynucleotide encoding the VHof the antibody of the invention, the VL of the antibody of theinvention, the heavy chain of the antibody of the invention or the lightchain of the antibody of the invention.

The invention also provides an isolated polynucleotide encoding the VH,the VL, or the VH and the VL of the antagonistic antibody specificallybinding PD-1 of the invention.

The invention also provides an isolated polynucleotide encoding the VHof SEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 63 or 64.

The invention also provides an isolated polynucleotide encoding the VLof SEQ ID NOs: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 or65.

The invention also provides an isolated polynucleotide comprising thepolynucleotide sequence of SEQ ID NOs: 196, 197, 198, 199, 200, 201, 202or 203.

The invention also provides an isolated polynucleotide encoding the VH,the VL, or the VH and the VL of the antagonistic antibody specificallybinding TIM-3 of the invention.

The invention also provides an isolated polynucleotide encoding the VHof SEQ ID NOs: 145, 146, 147, 148, 149, 150, 151, 152, 153, 154 or 172.

The invention also provides an isolated polynucleotide encoding the VLof SEQ ID NOs: 155, 156, 157, 158, 159, 160, 161, 162, 163 or 173.

The invention also provides an isolated polynucleotide comprising thepolynucleotide sequence of SEQ ID NOs: 204, 205, 206, 207, 208, 209, 210or 211.

The invention also provides an isolated polynucleotide encoding the HC1,the LC1, the HC2 or the LC2 of the antagonistic bispecific PD-1/TIM-3antibody of the invention.

The invention also provides an isolated polynucleotide encoding the HC1of SEQ ID NOs: 186, 187, 241, 242 or 243.

The invention also provides an isolated polynucleotide encoding the LC1of SEQ ID NOs: 188 or 189.

The invention also provides an isolated polynucleotide encoding the HC2of SEQ ID NOs: 190, 191, 192, 244, 245, 246, 247 or 248.

The invention also provides an isolated polynucleotide encoding the LC2of SEQ ID NOs: 193, 194 or 195.

The invention also provides an isolated polynucleotide comprising thepolynucleotide sequence of SEQ ID NOs: 253, 254, 255, 256, 257, 258, 259and 260.

(PD1H170) SEQ ID NO: 196CAGGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAACCGGGCAGCAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCGGCACCTTTAGCAGCTATGCGATTAGCTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATGGGCGGCATTATTCCGATTTTTGACACCGCGAACTATGCGCAGAAATTTCAGGGCCGCGTGACCATTACCGCGGATGAAAGCACCAGCACCGCGTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACCGCGGTGTATTATTGCGCGCGCCCTGGTCTCGCTGCGGCTTATGATACTGGTTCCTTGGACTATTGGGGCCAGGGCAC CCTGGTGACCGTGAGCAGC(PD1L148) SEQ ID NO: 197GAAATTGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGAGCCCGGGCGAACGCGCGACCCTGAGCTGCCGCGCGAGCCAGAGCGTTCGCTCCTACCTGGCGTGGTATCAGCAGAAACCGGGCCAGGCGCCGCGCCTGCTGATCTACGACGCGAGCAATCGTGCGACCGGCATTCCGGCGCGCTTTAGCGGCTCCGGTAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGGAACCGGAAGATTTTGCGGTGTATTATTGCCAGCAACGTAATTATTGGCCGCTGACCTTTGGCCAG GGCACCAAAGTGGAAATTAAA(PD1H129) SEQ ID NO: 198GAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCGCCTTCAGCAGATACGACATGAGCTGGGTGCGCCAGGCCCCTGGCAAAGGACTGGAAAGCGTGGCCTACATCTCTGGCGGAGGCGCCAACACCTACTACCTGGACAACGTGAAGGGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTATTGCGCCTCCCCCTACCTGAGCTACTTCGACGTGTGGGGCCAGGGCACACTCGTGACCGTGTCATC T (PD1L62)SEQ ID NO: 199 GAGATCGTGATGACCCAGAGCCCTGCCACCCTGTCCGTGTCTCCAGGCGAAAGAGCCACCCTGAGCTGCAGAGCCAGCCAGAGCCTGAGCGACTACCTGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCAAGTCTGCCAGCCAGTCCATCAGCGGCATCCCCGCCAGATTTTCTGGCAGCGGCTCCGGCACCGAGTTCACCCTGACAATCAGCAGCCTGCAGAGCGAGGACTTCGCCGTGTACTACTGCCAGAACGGCCACAGCTTCCCTTACACCTTCGGCCAG GGCACCAAGCTGGAAATCAAG(PD1H163) SEQ ID NO: 200CAGGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAACCGGGCAGCAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCGGCACCTTCAAGTCCTATGTGATTCATTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATGGGCGGTATTATCCCAATTTTTGGCACCGCCAATTATGCGCAGAAATTTCAGGGCCGCGTGACCATTACCGCTGATGAAAGCACCAGCACCGCGTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACCGCGGTGTATTATTGCGCGCGCGGTTATGTGCGGGCTACGGGCATGTTGGACTATTGGGGCCAGGGCACCC TGGTGACCGTGAGCAGC(PD1L185) SEQ ID NO: 201GAAATTGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGAGCCCGGGCGAACGCGCGACCCTGAGCTGCCGCGCGAGCCAGAGCGTTAGCAATTATCTGGCGTGGTATCAGCAGAAACCGGGCCAGGCGCCGCGCCTGCTGATCTACGACGCCAGCAATCGCGCGACCGGCATTCCGGCGCGCTTTAGCGGCTCCGGTAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGGAACCGGAAGATTTTGCGGTGTATTATTGCCAGCAACGTGCATATTGGCCGCTGACCTTTGGCCAGGGCACCAAAGTGGAAATTAAA (PD1H164) SEQ ID NO: 202CAGGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAACCGGGCAGCAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCGGCACCTTCAGCGATTATGTGATTTCCTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATGGGCGGTATTATCCCGATTTACGGGACCGCTAACTATGCGCAGAAATTTCAGGGCCGCGTGACCATTACCGCTGATGAAAGCACCAGCACCGCGTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACCGCGGTGTATTATTGCGCGCGCGGTACCCTCGACCGGACCGGGCATTTGGACTATTGGGGCCAGGGCACCC TGGTGACCGTGAGCAGC(PD1L86) SEQ ID NO: 203GAAATTGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGAGCCCGGGCGAACGCGCGACCCTGAGCTGCCGCGCGAGCCAGAGCGTCTCCTCCTACCTTGCGTGGTATCAGCAGAAACCGGGCCAGGCGCCGCGCCTGCTGATCCACGACGCCTCTACGCGTGCGACCGGCATTCCGGCGCGCTTTAGCGGCTCCGGTAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGGAACCGGAAGATTTTGCGGTGTATTATTGCCAGCAACGTAATTATTGGCCGCTCACCTTTGGCCAGGGCACCAAAGTGGAAATTAAA (TM3H24) SEQ ID NO: 204GAAGTGCAGCTGCTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCAAGCGGCTTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGAGCGCGATTAGCGGCAGCGGCGGCAGCACCTATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACAGCAAAAACACCCTGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCGCGAAATCCCCGTACGCGCCCTTGGACTATTGGGGCCAGGGCACCCTGGTGACCG TGAGCAGC (TM3L33)SEQ ID NO: 205 GAAATTGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGAGCCCGGGCGAACGCGCGACCCTTAGCTGCCGTGCAAGTCAGAGTGTGAACGACTACCTGGCGTGGTATCAGCAGAAACCGGGCCAGGCGCCGCGCCTGCTGATTTATGATGCGAGCAACCGCGCGACCGGCATTCCGGCGCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGGAACCGGAAGATTTTGCGGTGTATTATTGCCAGCAGGGTGGTCACGCGCCGATCACCTTTGGCCAGGGCACCAAAGTGGAAATTAAA (TM3H162) SEQ ID NO: 206GAAGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAGCCTGGCGAGAGCCTGAAGATCAGCTGCAAGGGCAGCGGCTACAGCTTCACCAGCTACTGGATGCAGTGGGTGCGCCAGATGCCTGGCAAGGGCCTGGAATGGATGGGCGCCATCTATCCCGGCGACGGCGACATCAGATACACCCAGAACTTCAAGGGCCAAGTGACCATCAGCGCCGACAAGAGCATCAGCACCGCCTACCTGCAGTGGTCCAGCCTGAAGGCCAGCGACACCGCCATGTACTACTGTGCCAGATGGGAGAAGTCCACCACCGTGGTGCAGCGGAACTACTTCGACTACTGGGGCCAGGGCACCACAGTGACCGTGTCTAGT (TM3L85) SEQ ID NO: 207GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACATGCAAGGCCAGCGAGAACGTGGGCACCTTCGTGTCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCAACAGATACACCGGCGTGCCCAGCAGATTCAGCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCTCTAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCGGCCAGAGCTACAGCTACCCCACCTTTGGCCAGGGCACCAAGCTGGAAATCAAG (TM3H21) SEQ ID NO: 208GAAGTGCAGCTGCTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTACCTTTAGCAACTATTGGATGAGCTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGAGCGCGATTAGCGGCAGCGGCGGCAGCACCTATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACAGCAAAAACACCCTGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCGCGAAAGATCATTGGGATCCCAATTTTTTGGACTATTGGGGCCAGGGCACCCTGG TGACCGTGAGCAGC (PH9L1)SEQ ID NO: 209 GAAATTGTGCTGACCCAGAGCCCGGGCACCCTGAGCCTGAGCCCGGGCGAACGCGCGACCCTGAGCTGCCGCGCGAGCCAGAGCGTGAGCAGCAGCTATCTGGCGTGGTATCAGCAGAAACCGGGCCAGGCGCCGCGCCTGCTGATTTATGGCGCGAGCAGCCGCGCGACCGGCATTCCGGATCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCCGCCTGGAACCGGAAGATTTTGCGGTGTATTATTGCCAGCAGTATGGCAGCAGCCCGCTGACCTTTGGCCAGGGCACCAAAGTGGAAATTAAA (TM3H65) SEQ ID NO: 210GAAGTGCAGCTGCTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTACCTTTAGCGACTATTGGATGAGCTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGAGCGTGATCAAGTATAGCGGTGGCTCCAAATATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACAGCAAAAACACCCTGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCGCGAAAGAGCTGGAGGGGGTGTTCGACTATTGGGGCCAGGGCACCCTGGTGACCG TGAGCAGC (TM3L12)SEQ ID NO: 211 GAAATTGTGCTGACCCAGAGCCCGGGCACCCTGAGCCTGAGCCCGGGCGAACGCGCGACCCTGAGCTGCCGCGCGAGCCAGAGCGTTAGCAATAGCACTCTGGCGTGGTATCAGCAGAAACCGGGCCAGGCGCCGCGCCTGCTGATTTATACTGCGAGCAGCCGCGCGACCGGCATTCCGGATCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCCGCCTGGAACCGGAAGATTTTGCGGTGTATTATTGCCAGCAGTCTTACACATCTCCGTGGACTTTTGGCCAGGGCACCAAAGTGGAAATTAAA

The polynucleotide sequences encoding the VH or the VL or anantigen-binding fragment thereof of the antibodies of the invention, orthe heavy chain and the light chain of the antibodies of the inventionmay be operably linked to one or more regulatory elements, such as apromoter or enhancer, that allow expression of the nucleotide sequencein the intended host cell. The polynucleotide may be a cDNA.

The invention also provides a vector comprising the polynucleotide ofthe invention. Such vectors may be plasmid vectors, viral vectors,vectors for baculovirus expression, transposon based vectors or anyother vector suitable for introduction of the synthetic polynucleotideof the invention into a given organism or genetic background by anymeans. For example, polynucleotides encoding light and/or heavy chainvariable regions of the antibodies of the invention, optionally linkedto constant regions, are inserted into expression vectors. The lightand/or heavy chains may be cloned in the same or different expressionvectors. The DNA segments encoding immunoglobulin chains may be operablylinked to control sequences in the expression vector(s) that ensure theexpression of immunoglobulin polypeptides. Such control sequencesinclude signal sequences, promoters (e.g. naturally associated orheterologous promoters), enhancer elements, and transcriptiontermination sequences, and are chosen to be compatible with the hostcell chosen to express the antibody. Once the vector has beenincorporated into the appropriate host, the host is maintained underconditions suitable for high level expression of the proteins encoded bythe incorporated polynucleotides.

In some embodiments, the vector comprises the polynucleotide of SEQ IDNO: 196 and 197.

In some embodiments, the vector comprises the polynucleotide of SEQ IDNO: 198 and 199.

In some embodiments, the vector comprises the polynucleotide of SEQ IDNO: 200 and 201.

In some embodiments, the vector comprises the polynucleotide of SEQ IDNO: 202 and 203.

In some embodiments, the vector comprises the polynucleotide of SEQ IDNO: 204 and 205.

In some embodiments, the vector comprises the polynucleotide of SEQ IDNO: 206 and 207.

In some embodiments, the vector comprises the polynucleotide of SEQ IDNO: 208 and 209.

In some embodiments, the vector comprises the polynucleotide of SEQ IDNO: 210 and 211.

In some embodiments, the vector comprises the polynucleotide of SEQ IDNO: 253 and 254.

In some embodiments, the vector comprises the polynucleotide of SEQ IDNO: 255 and 256.

In some embodiments, the vector comprises the polynucleotide of SEQ IDNO: 257 and 258.

In some embodiments, the vector comprises the polynucleotide of SEQ IDNO: 259 and 260.

Suitable expression vectors are typically replicable in the hostorganisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors contain selection markerssuch as ampicillin-resistance, hygromycin-resistance, tetracyclineresistance, kanamycin resistance or neomycin resistance to permitdetection of those cells transformed with the desired DNA sequences.

Suitable promoter and enhancer elements are known in the art. Forexpression in a eukaryotic cell, exemplary promoters include lightand/or heavy chain immunoglobulin gene promoter and enhancer elements;cytomegalovirus immediate early promoter; herpes simplex virus thymidinekinase promoter; early and late SV40 promoters; promoter present in longterminal repeats from a retrovirus; mouse metallothionein-I promoter;and various known tissue specific promoters. Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art.

Exemplary vectors that may be used are Bacterial: pBs, phagescript,PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a(Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3,pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo,pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL(Pharmacia), pEE6.4 (Lonza) and pEE12.4 (Lonza).

The invention also provides a host cell comprising one or more vectorsof the invention. “Host cell” refers to a cell into which a vector hasbeen introduced. It is understood that the term host cell is intended torefer not only to the particular subject cell but to the progeny of sucha cell, and also to a stable cell line generated from the particularsubject cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not be identical to the parent cell, but are still includedwithin the scope of the term “host cell” as used herein. Such host cellsmay be eukaryotic cells, prokaryotic cells, plant cells or archealcells. Escherichia coli, bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species are examples of prokaryotic host cells. Othermicrobes, such as yeast, are also useful for expression. Saccharomyces(for example, S. cerevisiae) and Pichia are examples of suitable yeasthost cells. Exemplary eukaryotic cells may be of mammalian, insect,avian or other animal origins. Mammalian eukaryotic cells includeimmortalized cell lines such as hybridomas or myeloma cell lines such asSP2/0 (American Type Culture Collection (ATCC), Manassas, Va.,CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury,Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCCCRL-1580) murine cell lines. An exemplary human myeloma cell line isU266 (ATTC CRL-TIB-196). Other useful cell lines include those derivedfrom Chinese Hamster Ovary (CHO) cells such as CHOK1SV (Lonza Biologics,Walkersville, Md.), Potelligent® CHOK2SV (Lonza), CHO-K1 (ATCC CRL-61)or DG44.

The invention also provides a method of producing an antibody of theinvention comprising culturing the host cell of the invention inconditions that the antibody is expressed, and recovering the antibodyproduced by the host cell. Methods of making antibodies and purifyingthem are well known in the art. Once synthesized (either chemically orrecombinantly), the whole antibodies, their dimers, individual lightand/or heavy chains, or other antibody fragments such as VH and/or VL,may be purified according to standard procedures, including ammoniumsulfate precipitation, affinity columns, column chromatography, highperformance liquid chromatography (HPLC) purification, gelelectrophoresis, and the like (see generally Scopes, ProteinPurification (Springer-Verlag, N.Y., (1982)). A subject antibody may besubstantially pure, for example, at least about 80% to 85% pure, atleast about 85% to 90% pure, at least about 90% to 95% pure, or at leastabout 98% to 99%, or more, pure, for example, free from contaminantssuch as cell debris, macromolecules, etc. other than the subjectantibody.

The polynucleotide sequences of the invention may be incorporated intovectors using standard molecular biology methods. Host celltransformation, culture, antibody expression and purification are doneusing well known methods. Another embodiment of the invention is amethod of producing the antagonistic antibody specifically binding PD-1of the invention, comprising:

-   -   incorporating the first polynucleotide encoding the VH of the        antibody and the second polynucleotide encoding the VL of the        antibody into an expression vector;    -   transforming a host cell with the expression vector;    -   culturing the host cell in culture medium under conditions        wherein the VL and the VH are expressed and form the antibody;        and    -   recovering the antibody from the host cell or culture medium.

Another embodiment of the invention described herein is a method ofproducing the antagonistic antibody specifically binding TIM-3 of theinvention, comprising:

-   -   incorporating the first polynucleotide encoding the VH of the        antibody and the second polynucleotide encoding the VL of the        antibody into an expression vector;    -   transforming a host cell with the expression vector;    -   culturing the host cell in culture medium under conditions        wherein the VL and the VH are expressed and form the antibody;        and    -   recovering the antibody from the host cell or culture medium.

The polynucleotides encoding certain VH or VL sequences of the inventiondescribed herein, and in some embodiments of each and every one of thenumbered embodiments listed below, may be incorporated into vectorsusing standard molecular biology methods. Host cell transformation,culture, antibody expression and purification are done using well knownmethods.

Pharmaceutical Compositions/Administration

The invention provides pharmaceutical compositions comprising theantibodies of the invention and a pharmaceutically acceptable carrier.For therapeutic use, the antibodies of the invention may be prepared aspharmaceutical compositions containing an effective amount of theantibody as an active ingredient in a pharmaceutically acceptablecarrier. “Carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the antibody of the invention is administered. Such vehiclesmay be liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3%glycine may be used. These solutions are sterile and generally free ofparticulate matter. They may be sterilized by conventional, well-knownsterilization techniques (e.g., filtration). The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, stabilizing, thickening, lubricating and coloring agents, etc.The concentration of the antibodies of the invention in suchpharmaceutical formulation may vary, from less than about 0.5%, usuallyto at least about 1% to as much as 15 or 20% by weight and may beselected primarily based on required dose, fluid volumes, viscosities,etc., according to the particular mode of administration selected.Suitable vehicles and formulations, inclusive of other human proteins,e.g., human serum albumin, are described, for example, in e.g.Remington: The Science and Practice of Pharmacy, 21^(st) Edition, Troy,D. B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.

The mode of administration for therapeutic use of the antibodies of theinvention may be any suitable route that delivers the antibody to thehost, such as parenteral administration, e.g., intradermal,intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary,transmucosal (oral, intranasal, intravaginal, rectal), using aformulation in a tablet, capsule, solution, powder, gel, particle; andcontained in a syringe, an implanted device, osmotic pump, cartridge,micropump; or other means appreciated by the skilled artisan, as wellknown in the art. Site specific administration may be achieved by forexample intratumoral, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracerebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intracardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravascular, intravesical,intralesional, vaginal, rectal, buccal, sublingual, intranasal, ortransdermal delivery.

The antibodies of the invention may be administered to a subject by anysuitable route, for example parentally by intravenous (i.v.) infusion orbolus injection, intramuscularly or subcutaneously or intraperitoneally.i.v. infusion may be given over for example 15, 30, 60, 90, 120, 180, or240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

The dose given to a subject is sufficient to alleviate or at leastpartially arrest the disease being treated (“therapeutically effectiveamount”) and may be sometimes 0.005 mg to about 100 mg/kg, e.g. about0.05 mg to about 30 mg/kg or about 5 mg to about 25 mg/kg, or about 4mg/kg, about 8 mg/kg, about 16 mg/kg or about 24 mg/kg, or for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but may even higher, forexample about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50,60, 70, 80, 90 or 100 mg/kg.

A fixed unit dose may also be given, for example, 50, 100, 200, 500 or1000 mg, or the dose may be based on the patient's surface area, e.g.,500, 400, 300, 250, 200, or 100 mg/m². Usually between 1 and 8 doses,(e.g., 1, 2, 3, 4, 5, 6, 7 or 8) may be administered to treat thepatient, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more dosesmay be given.

The administration of the antibodies of the invention may be repeatedafter one day, two days, three days, four days, five days, six days, oneweek, two weeks, three weeks, one month, five weeks, six weeks, sevenweeks, two months, three months, four months, five months, six months orlonger. Repeated courses of treatment are also possible, as is chronicadministration. The repeated administration may be at the same dose orat a different dose. For example, the antibodies of the invention may beadministered at 8 mg/kg or at 16 mg/kg at weekly interval for 8 weeks,followed by administration at 8 mg/kg or at 16 mg/kg every two weeks foran additional 16 weeks, followed by administration at 8 mg/kg or at 16mg/kg every four weeks by intravenous infusion.

For example, the antibodies of the invention may be provided as a dailydosage in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1,1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively,at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 after initiation of treatment, or any combinationthereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2hours, or any combination thereof.

The antibodies of the invention, may also be administeredprophylactically in order to reduce the risk of developing cancer, delaythe onset of the occurrence of an event in cancer progression, and/orreduce the risk of recurrence when a cancer is in remission.

The antibodies of the invention may be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional protein preparations andwell known lyophilization and reconstitution techniques can be employed.

Methods and Uses

The antibodies of the invention have in vitro and in vivo diagnostic, aswell as therapeutic and prophylactic utilities. For example, theantibodies of the invention may be administered to cells in culture, invitro or ex vivo, or to a subject to treat, prevent, and/or diagnose avariety of disorders, such as cancers and infectious disorders.

The invention provides a method of modifying an immune response in asubject comprising administering to the subject the antibody of theinvention for a time sufficient to modify the immune response.

In some embodiments, the immune response is enhanced, stimulated orupregulated.

In some embodiments described herein, the subject is a human patient.

In some embodiments described herein, the subject is a human patient inneed of enhancement of the immune response.

In some embodiments, the subject is immunocompromised.

In some embodiments, the subject is at risk of being immunocompromised.Immunocompromised subject may be undergoing, or has undergone achemotherapeutic or radiation therapy.

In some embodiment, the subject is or is at risk of beingimmunocompromised as a result of an infection.

The antibodies of the invention are suitable for treating a subjecthaving a disorder that may be treated by augmenting T-cell mediatedimmune responses.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention described herein is PD1B114,PD1B149, PD1B160, PD1B162, PD1B164, PD1B11, PD1B183, PD1B184, PD1B185,PD1B187, PD1B71, PD1B177, PD1B70, PD1B175, PD1B194, PD1B195, PD1B196,PD1B197, PD1B198, PD1B199, PD1B200, PD1B201, PD1B243, PD1B244, PD1B131or PD1B132. The VH and the VL amino acid sequences of these antibodiesare shown in Table 2.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 used in the methods of the invention described herein is TM3B103,TM3B105, TM3B109, TM3B108, TM3B113, TM3B189, TM3B190, TM3B193, TM3B195,TM3B196 or TM3B291. The VH and the VL amino acid sequences of theseantibodies are shown in Table 3.

In some embodiments, the bispecific PD-1/TIM-3 antibody used in themethods of the invention is PTBB14, PTBB15, PTBB16, PTBB17, PTBB24,PTBB30, PTBB27, PTBB28, PTBB18, PTBB20 or PTBB21. The HC1, the LC1, theHC2 and the LC2 amino acid sequences of these antibodies are shown inTable 41 and Table 42.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 41and the VL of SEQ ID NO: 49.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 41and the VL of SEQ ID NO: 50.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 42and the VL of SEQ ID NO: 51.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 42and the VL of SEQ ID NO: 52.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 42and the VL of SEQ ID NO: 53.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 43and the VL of SEQ ID NO: 49.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 43and the VL of SEQ ID NO: 54.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 43and the VL of SEQ ID NO: 50.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 43and the VL of SEQ ID NO: 55.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 43and the VL of SEQ ID NO: 56.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 43and the VL of SEQ ID NO: 57.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 44and the VL of SEQ ID NO: 49.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 45and the VL of SEQ ID NO: 49.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 46and the VL of SEQ ID NO: 49.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 47and the VL of SEQ ID NO: 49.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 48and the VL of SEQ ID NO: 53.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 48and the VL of SEQ ID NO: 52.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 48and the VL of SEQ ID NO: 56.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 47and the VL of SEQ ID NO: 58.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 47and the VL of SEQ ID NO: 59.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 45and the VL of SEQ ID NO: 60.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 45and the VL of SEQ ID NO: 61.

In some embodiments, the antagonistic antibody specifically binding PD-1used in the methods of the invention comprises the VH of SEQ ID NO: 45and the VL of SEQ ID NO: 62.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 used in the methods of the invention comprises the VH of SEQ IDNO: 145 and the VL of SEQ ID NO: 155.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 used in the methods of the invention comprises the VH of SEQ IDNO: 146 and the VL of SEQ ID NO: 156.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 used in the methods of the invention comprises the VH of SEQ IDNO: 148 and the VL of SEQ ID NO: 157.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 used in the methods of the invention comprises the VH of SEQ IDNO: 147 and the VL of SEQ ID NO: 155.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 used in the methods of the invention comprises the VH of SEQ IDNO: 149 and the VL of SEQ ID NO: 158.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 used in the methods of the invention comprises the VH of SEQ IDNO: 150 and the VL of SEQ ID NO: 159.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 used in the methods of the invention comprises the VH of SEQ IDNO: 151 and the VL of SEQ ID NO: 160.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 used in the methods of the invention comprises the VH of SEQ IDNO: 152 and the VL of SEQ ID NO: 161.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 used in the methods of the invention comprises the VH of SEQ IDNO: 153 and the VL of SEQ ID NO: 162.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 used in the methods of the invention comprises the VH of SEQ IDNO: 154 and the VL of SEQ ID NO: 163.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 used in the methods of the invention comprises the VH of SEQ IDNO: 172 and the VL of SEQ ID NO: 173.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibodycomprising a first domain specifically binding PD-1 and a second domainspecifically binding TIM-3 used in the methods of the inventioncomprises the VH of SEQ ID NO: 48 and the VL of SEQ ID NO: 56 in thefirst domain, and the VH of SEQ ID NO: 153 and the VL of SEQ ID NO: 162in the second domain.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibodycomprising a first domain specifically binding PD-1 and a second domainspecifically binding TIM-3 used in the methods of the inventioncomprises the VH of SEQ ID NO: 48 and the VL of SEQ ID NO: 56 in thefirst domain, and the VH of SEQ ID NO: 146 and the VL of SEQ ID NO: 156in the second domain.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibodycomprising a first domain specifically binding PD-1 and a second domainspecifically binding TIM-3 used in the methods of the inventioncomprises the VH of SEQ ID NO: 64 and the VL of SEQ ID NO: 65 in thefirst domain, and the VH of SEQ ID NO: 153 and the VL of SEQ ID NO: 162in the second domain.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibodycomprising a first domain specifically binding PD-1 and a second domainspecifically binding TIM-3 used in the methods of the invention,comprises the VH of SEQ ID NO: 64 and the VL of SEQ ID NO: 65 in thefirst domain, and the VH of SEQ ID NO: 146 and the VL of SEQ ID NO: 156in the second domain.

In some embodiments, the antagonistic bispecific PD-1/TIM-3 antibodycomprising a first domain specifically binding PD-1 and a second domainspecifically binding TIM-3 used in the methods of the inventioncomprises the VH of SEQ ID NO: 48 and the VL of SEQ ID NO: 56 in thefirst domain, and the VH of SEQ ID NO: 172 and the VL of SEQ ID NO: 173in the second domain.

Cancer

Blockade of PD-1 may enhance an immune response to cancerous cells in asubject. The ligand for PD-1, PD-L1, is abundantly expressed in avariety of human cancers (Dong et al., (2002) Nat Med 8:787-9). Theinteraction between PD-1 and PD-L1 can result in a decrease in tumorinfiltrating lymphocytes, a decrease in T-cell receptor mediatedproliferation, and/or immune evasion by the cancerous cells (Dong etal., (2003) J Mol Med 81:281-7; Blank et al., (2005) Cancer ImmunolImmunother 54:307-314; Konishi et al., (2004) Clin Cancer Res10:5094-100) Immune suppression may be reversed by inhibiting the localinteraction of PD-1 to PD-L1; the effect is additive when theinteraction of PD-1 to the second PD-1 ligand, PD-L2, is blocked as well(Iwai et al., (2002) PorcNatl Acad Sci 99:12293-7; Brown et al., (2003)J Immunol 170:1257-66). Thus, inhibition of PD-1 may result inaugmenting an immune response.

TIM-3 is a coinhibitory protein expressed on activated T helper 1 (Th1)CD4⁺ and cytotoxic CD8⁺ T cells that secrete IFN-γ. TIM-3 isco-expressed on PD-1+ exhausted T cells as shown in preclinical modelsof cancer and viral exhaustion. Co-blockade of these pathways mayrestore effector T cell function (e.g., IFN-γ secretion, proliferation)in several models as well as human PBMCs derived from metastaticmelanoma patients and patients with HIV or HCV. TIM-3 is also enrichedon Foxp3+ regulatory T cells and Tregs co-expressing TIM-3, LAG3 andCTLA4 have been shown to be highly efficient suppressors of effector Tcells (Teff) (Galuton et al., (2014) Eur J Immunol 44(9):2703-11). TIM-3expression has been correlated with poorer prognosis in NSCLC (Zhuang etal., (2012) Am J Clin Pathol 137(6):978-85). Lymphocytes from tumortissues of ovarian, colorectal, cervical and hepatocellular carcinomapatients exhibit higher proportion of TIM-3⁺ CD4 T cells, which cellshave impaired capacity to produce ILF-γ (Yan et al., (2013) PLoS One8(3):e58006).

The invention also provides a method of inhibiting growth of tumor cellsin a subject, comprising administering to the subject a therapeuticallyeffective amount of the antagonistic antibody specifically binding PD-1of the invention for a time sufficient to inhibit growth of tumor cells.

The invention also provides a method of inhibiting growth of tumor cellsin a subject, comprising administering to the subject a therapeuticallyeffective amount of the antagonistic antibody specifically binding TIM-3of the invention for a time sufficient to inhibit growth of tumor cells.

The invention also provides a method of inhibiting growth of tumor cellsin a subject, comprising administering to the subject a therapeuticallyeffective amount of the antagonistic bispecific PD-1/TIM-3 antibody ofthe invention for a time sufficient to inhibit growth of tumor cells.

The invention also provides a method of treating a cancer byadministering to the subject in need thereof a therapeutically effectiveamount of the antagonistic antibody specifically binding PD-1 of theinvention for a time sufficient to treat the cancer.

The invention also provides a method of treating a cancer byadministering to the subject in need thereof a therapeutically effectiveamount of the antagonistic antibody specifically binding TIM-3 of theinvention for a time sufficient to treat the cancer.

The invention also provides a method of treating a cancer byadministering to the subject in need thereof a therapeutically effectiveamount of the bispecific PD-1/TIM-3 antibody of the invention for a timesufficient to treat the cancer.

Exemplary antibodies that may be used are antagonistic antibodiesspecifically binding PD-1, antagonistic antibodies specifically bindingTIM-3, and antagonistic bispecific PD-1/TIM-3 antibodies PD1B114,PD1B149, PD1B160, PD1B162, PD1B164, PD1B11, PD1B183, PD1B184, PD1B185,PD1B187, PD1B71, PD1B177, PD1B70, PD1B175, PD1B194, PD1B195, PD1B196,PD1B197, PD1B198, PD1B199, PD1B200, PD1B201, TM3B103, TM3B105, TM3B109,TM3B108, TM3B113, TM3B189, TM3B190, TM3B193, TM3B195, TM3B196, TM3B291,PTBB14, PTBB15, PTBB16, PTBB17, PTBB24, PTBB30, PTBB27, PTBB28, PTBB18,PTBB20 and PTBB21 having the VH and the VL amino acid sequence andcharacteristics as described herein.

Cancer may be a hyperproliferative condition or disorder, a solid tumor,a hematological malignancy, a soft tissue tumor, or a metastatic lesion.

“Cancer” is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathology type or stage of invasiveness.Examples of cancers include solid tumors, hematological malignancies,soft tissue tumors, and metastatic lesions. Exemplary solid tumorsinclude malignancies, e.g., sarcomas, and carcinomas (includingadenocarcinomas and squamous cell carcinomas) of the various organsystems, such as those affecting liver, lung, breast, lymphoid,gastrointestinal (e.g., colon), genitourinary tract (e.g., renal,urothelial cells), prostate and pharynx. Adenocarcinomas includemalignancies such as most colon cancers, a rectal cancer, a renal-cellcarcinoma, a liver cancer, a non-small cell carcinoma of the lung, acancer of the small intestine and a cancer of the esophagus. Squamouscell carcinomas include malignancies, e.g., in the lung, esophagus,skin, head and neck region, oral cavity, anus, and cervix.

In some embodiments, the cancer is a melanoma.

Metastatic lesions of the aforementioned cancers may also be treated orprevented using the methods and antibodies of the invention describedherein.

Exemplary cancers whose growth may be inhibited or reduced using theantibodies of the invention include cancers that may be responsive toimmunotherapy. Exemplary such cancers include a melanoma, a renalcancer, a prostate cancer, a breast cancer, a colon cancer, agastrointestinal cancer, a stomach cancer, an esophageal cancer, a lungcancer, a metastatic malignant melanoma, a clear cell carcinoma, ahormone refractory prostate adenocarcinoma, a non-small cell lung canceror cancer of the head and neck. Refractory or recurrent malignancies maybe treated using the antibodies of the invention described herein.

Exemplary other cancers that may be treated with the antibodies of theinvention ae an anal cancer, a basal cell carcinoma, a biliary tractcancer, a bladder cancer, a bone cancer, brain and CNS cancers, acarcinoma of the fallopian tubes, carcinoma of the vagina, a carcinomaof the vulva, a cutaneous or intraocular malignant melanoma, aastro-esophageal cancer, a testicular cancer, an ovarian cancer, apancreatic cancer, a rectal cancer, an uterine cancer, a primary CNSlymphoma; a neoplasm of the central nervous system (CNS), a cervicalcancer, a choriocarcinoma, a rectum cancer, a connective tissue cancer,a cancer of the digestive system, an endometrial cancer, an eye cancer;an intra-epithelial neoplasm, a kidney cancer, a larynx cancer, a livercancer; a small cell lung cancer, a neuroblastoma, an oral cavity cancer(e.g., lip, tongue, mouth, and pharynx), a nasopharyngeal cancer, aretinoblastoma, a rhabdomyosarcoma, a cancer of the respiratory system,a sarcoma, a thyroid cancer, a cancer of the urinary system, ahepatocarcinoma, a cancer of the anal region, a carcinoma of thefallopian tubes, a carcinoma of the vagina, a carcinoma of the vulva, acancer of the small intestine, a cancer of the endocrine system, acancer of the parathyroid gland, a cancer of the adrenal gland, asarcoma of soft tissue, a cancer of the urethra, a cancer of the penis,solid tumors of childhood, a tumor angiogenesis, a spinal axis tumor, abrain stem glioma, a pituitary adenoma, Kaposi's sarcoma, Merkel cellcancer, an epidermoid cancer, a squamous cell cancer, an environmentallyinduced cancers including those induced by asbestos, as well as othercarcinomas and sarcomas, and combinations of said cancers.

Exemplary hematological malignancies that may be treated with theantibodies of the invention include leukemias, lymphomas and myeloma,such as a precursor B-cell lymphoblastic leukemia/lymphoma and a B-cellnon-Hodgkin's lymphoma, an acute promyelocytic leukemia, an acutelymphoblastic leukemia (ALL), a B-cell chronic lymphocytic leukemia(CLL)/small lymphocytic lymphoma (SLL), a B-cell acute lymphocyticleukemia, a B-cell prolymphocytic leukemia, a lymphoplasmacyticlymphoma, a mantle cell lymphoma (MCL), a follicular lymphoma (FL),including low-grade, intermediate-grade and high-grade FL, a cutaneousfollicle center lymphoma, a marginal zone B-cell lymphoma (MALT type,nodal and splenic type), a hairy cell leukemia, a diffuse large B-celllymphoma (DLBCL), Burkitt's lymphoma (BL), a plasmacytoma, a multiplemyeloma (MM), a plasma cell leukemia, a post-transplantlymphoproliferative disorder, Waldenstrom's macroglobulinemia, plasmacell disorders, an anaplastic large-cell lymphoma (ALCL), a T-cell acutelymphocytic leukemia, a primary systemic amyloidosis (e.g. light chainamyloidosis), a pro-lymphocytic/myelocytic leukemia, an acute myeloidleukemia (AML), a chronic myeloid leukemia (CML), a large granularlymphocytic (LGL) leukemia, a NK-cell leukemia and Hodgkin's lymphoma.

“Plasma cell disorder” refers to disorders characterized by clonalplasma cells, and includes a multiple myeloma, a light chain amyloidosisand Waldenstrom's macroglobulinemia. Light chain amyloidosis andWaldenstrom's macroglobulinemia can arise independently from multiplemyeloma. They may also present simultaneously with multiple myeloma, anddevelop either before or after the development of multiple myeloma.

Exemplary B-cell non-Hodgkin's lymphomas are a lymphomatoidgranulomatosis, a primary effusion lymphoma, an intravascular largeB-cell lymphoma, a mediastinal large B-cell lymphoma, heavy chaindiseases (including γ, μ, and a disease), lymphomas induced by therapywith immunosuppressive agents, such as cyclosporine-induced lymphoma,and methotrexate-induced lymphoma.

Patients having cancer including metastatic cancer that express PD-L1may be treated with the antibodies of the invention. The cancer may be amelanoma, a renal cell carcinoma, a squamous non-small cell lung cancer(NSCLC), a non-squamous NSCLC, a colorectal cancer, acastration-resistant prostate cancer, an ovarian cancer, a gastriccancer, an adenocarcinoma (ACA), a squamous cell carcinoma (SCC), ahepatocellular carcinoma (HCC), a pancreatic carcinoma, a squamous cellcarcinoma of the head and neck, carcinomas of the esophagus,gastrointestinal tract and breast.

Patients having cancer that expresses TIM-3 may be treated with theantibodies of the invention. TIM-3-expressing cancers include a cervicalcancer, a lung cancer, a NSCLC, an acute myeloid leukemia (AML), adiffuse large B cell lymphoma (DLBCL), a melanoma, a renal cancer, arenal cell carcinoma (RCC), a kidney clear cell carcinoma, a kidneypapillary cell carcinoma, a metastatic renal cell carcinoma, a squamouscell carcinoma, an esophageal squamous cell carcinoma, a nasopharyngealcarcinoma, a colorectal cancer, a breast cancer (e.g., a breast cancerthat does not express one, two or all of estrogen receptor, progesteronereceptor, or Her2/neu, e.g., a triple negative breast cancer), amesothelioma, a hepatocellular carcinoma, and an ovarian cancer. TheTIM-3-expressing cancer may be a metastatic cancer.

In some embodiments, the subject has a solid tumor.

In some embodiments, the subject has a hematological malignancy.

In some embodiments, the solid tumor is a melanoma.

In some embodiments, the solid tumor is a lung cancer.

In some embodiments, the solid tumor is a non-small cell lung cancer(NSCLC).

In some embodiments, the solid tumor is a squamous non-small cell lungcancer (NSCLC).

In some embodiments, the solid tumor is a non-squamous NSCLC.

In some embodiments, the solid tumor is a lung adenocarcinoma.

In some embodiments, the solid tumor is a renal cell carcinoma (RCC).

In some embodiments, the solid tumor is a mesothelioma.

In some embodiments, the solid tumor is a nasopharyngeal carcinoma(NPC).

In some embodiments, the solid tumor is a colorectal cancer.

In some embodiments, the solid tumor is a prostate cancer.

In some embodiments, the solid tumor is castration-resistant prostatecancer.

In some embodiments, the solid tumor is a stomach cancer.

In some embodiments, the solid tumor is an ovarian cancer.

In some embodiments, the solid tumor is a gastric cancer.

In some embodiments, the solid tumor is a liver cancer.

In some embodiments, the solid tumor is pancreatic cancer.

In some embodiments, the solid tumor is a thyroid cancer.

In some embodiments, the solid tumor is a squamous cell carcinoma of thehead and neck.

In some embodiments, the solid tumor is a carcinomas of the esophagus orgastrointestinal tract.

In some embodiments, the solid tumor is a breast cancer.

In some embodiments, the solid tumor is a fallopian tube cancer.

In some embodiments, the solid tumor is a brain cancer.

In some embodiments, the solid tumor is an urethral cancer.

In some embodiments, the solid tumor is a genitourinary cancer.

In some embodiments, the solid tumor is an endometriosis.

In some embodiments, the solid tumor is a cervical cancer.

In some embodiments, the solid tumor is a metastatic lesion of thecancer.

In some embodiments, the hematological malignancy is a lymphoma, amyeloma or a leukemia.

In some embodiments, the hematological malignancy is a B cell lymphoma.

In some embodiments, the hematological malignancy is Burkitt's lymphoma.

In some embodiments, the hematological malignancy is Hodgkin's lymphoma.

In some embodiments, the hematological malignancy is a non-Hodgkin'slymphoma.

In some embodiments, the hematological malignancy is a myelodysplasticsyndrome.

In some embodiments, the hematological malignancy is an acute myeloidleukemia (AML).

In some embodiments, the hematological malignancy is a chronic myeloidleukemia (CML).

In some embodiments, the hematological malignancy is a chronicmyelomoncytic leukemia (CMML).

In some embodiments, the hematological malignancy is a multiple myeloma(MM).

In some embodiments, the hematological malignancy is a plasmacytoma.

In some embodiments, the subject has a tumor that expresses PD-L1.

In some embodiments, the subject has tumor-infiltrating T lymphocytes(TILs) in the tumor tissue.

In some embodiments, the subject has PD-1⁺TIM-3⁺ TILs in the tumortissue. In some embodiments, the subject has increased number of PD-1⁺TIM-3⁺ tumor-infiltrating T lymphocytes (TILs) in the tumor tissue.

“Increased number” refers to statistically significant increase in asubject when compared to a control. “Increased number” for examplerefers to statistically significant increase in the number of TILs in asubject (e.g. patient) pre- and post-treatment with a PD-1 antibody orother therapeutic.

In some embodiments, the subject has increased expression or activity ofinterferon-gamma (IFN-γ).

In some embodiments, the subject has been treated with an anti-PD-1antibody.

In some embodiments, the subject is refractory to treatment with theanti-PD-1 antibody.

In some embodiments, the subject has a relapsed tumor after treatmentwith the anti-PD-1 antibody.

In some embodiments, the subject has been treated with the anti-PD-1antibody comprising the VH of SEQ ID NO: 230 and the VL of SEQ ID NO:231 (e.g. KEYTRUDA® (pembrolizumab)).

In some embodiments, the subject has been treated with the anti-PD-1antibody comprising the VH of SEQ ID NO: 232 and the VL of SEQ ID NO:233 (e.g. OPDIVO® (nivolumab)).

In some embodiments, the subject is refractory to treatment with theanti-PD-1 antibody comprising the VH of SEQ ID NO: 230 and the VL of SEQID NO: 231 (e.g. KEYTRUDA® (pembrolizumab)).

In some embodiments, the subject is refractory to treatment with theanti-PD-1 antibody comprising the VH of SEQ ID NO: 232 and the VL of SEQID NO: 233 (e.g. OPDIVO® (nivolumab)).

In some embodiments, the subject has a relapsed tumor after treatmentwith the anti-PD-1 antibody comprising the VH of SEQ ID NO: 230 and theVL of SEQ ID NO: 231 (e.g. KEYTRUDA® (pembrolizumab).

In some embodiments, the subject has a relapsed tumor after treatmentwith the anti-PD-1 antibody comprising the VH of SEQ ID NO: 232 and theVL of SEQ ID NO: 233 (e.g. OPDIVO® (nivolumab)).

SEQ ID NO: 230 QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRD YRFDMGFDYWGQGTTVTVSSSEQ ID NO: 231 EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPL TFGGGTKVEIKSEQ ID NO: 232 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATND DYWGQGTLVTVSSSEQ ID NO: 233 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQ GTKVEIK

In some embodiments, the subject has been treated or is being treatedwith a PD-L1 antibody.

In some embodiments, the subject is refractory to treatment with thePD-L1 antibody.

In some embodiments, the subject has a relapsed tumor after treatmentwith the PD-L1 antibody.

In some embodiments, the subject is refractory or relapsed aftertreatment with the PD-L1 antibody durvalumab (MEDI-4736). Durvalumabcomprises the VH of SEQ ID NO: 234 and the VL of SEQ ID NO: 235.

SEQ ID NO: 234 EVQLVESGGG LVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS SEQ ID NO: 235EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQK PGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTF GQGTKVEIK

In some embodiments, the subject is refractory or relapsed aftertreatment with the PD-L1 antibody atezolizumab.

Atezolizumab comprises the VH of SEQ ID NO: 236 and the VL of SEQ ID NO:237.

SEQ ID NO: 236 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH WPGGFDYWGQGTLVTVSSSEQ ID NO: 237 DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIK

In some embodiments, the subject is refractory or relapsed aftertreatment with the PD-L1 antibody avelumab.

Avelumab comprises the VH of SEQ ID NO: 238 and the VL of SEQ ID NO:239.

SEQ ID NO: 238 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIK LGTVTTVDYWGQGTLVTVSSSEQ ID NO: 239 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRV FGTGTKVTVL

In some embodiments, the subject is refractory or relapsed aftertreatment with the PD-L1 antibody MDX-1105.

In some embodiments, the subject has been treated or is being treatedwith a PD-L2 antibody.

In some embodiments described herein, the subject is refractory totreatment with a PD-L2 antibody.

In some embodiments, the subject has a relapsed tumor after treatmentwith a PD-L2 antibody.

Various qualitative and/or quantitative methods may be used to determinerelapse or refractory nature of the disease. Symptoms that may beassociated with relapse or resistance are, for example, a decline orplateau of the well-being of the patient or re-establishment orworsening of various symptoms associated with solid tumors, and/or thespread of cancerous cells in the body from one location to other organs,tissues or cells.

TIM-3 expression was found herein to be elevated in CD8⁺ T cellsisolated from tumors after anti-PD-1 antibody treatment. Therefore,therapeutic administration of antagonistic antibodies specificallybinding TIM-3 or antagonistic bispecific PD-1/TIM-3 antibodies describedherein to a subject who has already received or is receiving anti-PD-1antibody therapy, is refractory to the anti-PD-1 antibody treatment orhas relapsed after or during the anti-PD-1 antibody treatment mayimprove the clinical outcome of the patients.

The invention also provides a method of treating a cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of the antagonistic antibody specifically binding TIM-3 of theinvention, wherein the subject is being treated or has been treated withan anti-PD-1 antibody.

In some embodiments, the antagonistic antibody specifically bindingTIM-3 comprises the VH of SEQ ID NO: 146 and the VL of SEQ ID NO: 156.

The invention also provides a method of treating a cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of the antagonistic antibody specifically binding TIM-3comprising the VH of SEQ ID NO: 146 and the VL of SEQ ID NO: 156,wherein the subject is being treat or has been treated with theanti-PD-1 antibody KEYTRUDA® (pembrolizumab) comprising the VH of SEQ IDNO: 230 and the VL of SEQ ID NO: 231.

The invention also provides a method of treating a cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of the antagonistic antibody specifically binding TIM-3comprising the VH of SEQ ID NO: 146 and the VL of SEQ ID NO: 156,wherein the subject is being treat or has been treated with theanti-PD-1 antibody OPDIVO® (nivolumab) comprising the VH of SEQ ID NO:232 and the VL of SEQ ID NO: 233.

The invention also provides a method of treating a cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of the antagonistic antibody specifically binding TIM-3 of theinvention, wherein the subject is being treated or has been treated withan anti-PD-L1 antibody.

The invention also provides a method of treating a cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of the antagonistic antibody specifically binding TIM-3 of theinvention, wherein the subject is being treated or has been treated withan anti-PD-L2 antibody.

The invention also provides a method of treating a cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of the antagonistic bispecific PD-1/TIM-3 antibody the invention,wherein the subject is being treated or has been treated with ananti-PD-1 antibody.

The invention also provides a method of treating a cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of the antagonistic bispecific PD-1/TIM-3 antibody the invention,wherein the subject is being treated or has been treated with ananti-PD-L1 antibody.

The invention also provides a method of treating a cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of the antagonistic bispecific PD-1/TIM-3 antibody the invention,wherein the subject is being treated or has been treated with ananti-PD-L2 antibody.

The invention also provides a method of treating a cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of the antagonistic antibody specifically binding PD-1 comprisingthe VH of SEQ ID NO: 48 and the VL of SEQ ID NO: 56 for a timesufficient to treat the cancer.

The invention also provides a method of treating a cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of the antagonistic antibody specifically binding PD-1 comprisingthe VH of SEQ ID NO: 64 and the VL of SEQ ID NO: 65 for a timesufficient to treat the cancer.

Any of the PD-1, TIM-3 or bispecific PD-1/TIM-3 antibodies of theinvention described herein may be used in the methods of the invention.

“Treat” or “treatment” refers to therapeutic treatment wherein theobject is to slow down (lessen) an undesired physiological change ordisease, such as the development or spread of tumor or tumor cells, orto provide a beneficial or desired clinical outcome during treatment.Beneficial or desired clinical outcomes include alleviation of symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, lack ofmetastasis, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” may also mean prolonging survival as comparedto expected survival if a subject was not receiving treatment. Those inneed of treatment include those subjects already with the undesiredphysiological change or diseases well as those subjects prone to havethe physiological change or disease.

A “therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve a desiredtherapeutic result. A therapeutically effective amount of the antibodyof the invention may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody of the invention to elicit a desired response in theindividual. Exemplary indicators of an effective therapeutic orcombination of therapeutics include, for example, improved well-being ofthe patient, reduction in a tumor burden, arrested or slowed growth of atumor, and/or absence of metastasis of cancer cells to other locationsin the body.

Combination Therapies for Cancer Treatment

The antibodies of the invention may be administered in combination witha second therapeutic agent.

The antibodies of the invention may be administered in combination withone, two, three, four, five or six additional therapeutic agents.

Any of the antagonistic antibodies specifically binding PD-1,antagonistic antibodies specifically binding TIM-3 or antagonisticbispecific PD-1/TIM-3 antibodies of the invention may be used incombination with a second therapeutic agent.

Any of the antagonistic antibodies specifically binding PD-1,antagonistic antibodies specifically binding TIM-3 or antagonisticbispecific PD-1/TIM-3 antibodies of the invention may be used incombination with one, two, three, four, five or six additionaltherapeutic agents.

“In combination with” refers to administering of the antibodies of theinvention and at least one second therapeutic agent concurrently assingle agents or sequentially as single agents in any order. In general,each agent will be administered at a dose and/or on a time scheduledetermined for that agent.

In some embodiments, the second therapeutic agent modulates activity ofa molecule involved in the cancer-immunity cycle, e.g. a moleculeinvolved in stimulatory or inhibitory pathways functioning in release ofcancer cell antigens, cancer antigen presentation, T cell priming andactivation, trafficking of T cells to tumors, infiltration of T cellsinto tumors, recognition of cancer cells by T cells, and killing ofcancer cells. The cancer-immunity cycle is described in Chen and Mellman(2013) Immunity 39:1-10. In some embodiments, the second therapeuticagend modulates activity of a molecule involved in regulation ofactivity of T regulatory cells (Treg), co-stimulatory or co-inhibitoryligands expressed on tumors, activating or inhibitory receptors onnatural killer (NK) cells, or immunosuppressive factors in the tumormicroenvironment. Combination cancer immunotherapies are described inManoney et al., (2015) Nature Reviews 14:561-584.

The second therapeutic agent typically enhances the activity ofstimulatory molecules and suppresses the activity of inhibitorymolecules, as is well known. Thus, “modulate” refers to the enhancementof immune response by the second therapeutic agent, wheatear the agentitself is agonist or antagonist of a specific molecule.

In some embodiments, the antibodies of the invention are administered incombination with an inhibitor of a T cell inhibitory molecule.

In some embodiments, the antibodies of the invention are administered incombination with an inhibitor of a T cell inhibitory molecule PD-1,PD-L1, PD-L2, VISTA, BTNL2, B7-H3, B7-H4, HVEM, HHLA2, CTLA-4, LAG-3,TIM-3, BTLA, CD160, CEACAM-1, LAIR1, TGFβ, IL-10, Siglec family protein,KIR, CD96, TIGIT, NKG2A, CD112, CD47, SIRPA or CD244.

In some embodiments, KIR is KIR2DL1, KIR2DL2 or KIR2DL3.

Inhibition of inhibitory molecules may be performed by inhibition at theDNA, RNA or protein level. In some embodiments, an inhibitory nucleicacid (e.g., a dsRNA, siRNA or shRNA) is used to inhibit expression ofthe inhibitory molecule.

In some embodiments, the inhibitor of the inhibitory molecule is asoluble ligand of the inhibitory molecule.

In some embodiments, the inhibitor of the inhibitory molecule is anantagonistic antibody specifically binding the inhibitory molecule.

In some embodiments, the inhibitor of the inhibitory molecule isCTLA-4-Fc or TIM-3-Fc fusion protein.

In some embodiments, the inhibitor of the inhibitory molecule is anantibody or an antibody fragment that binds PD-1, PD-L1, PD-L2, VISTA,BTNL2, B7-H3, B7-H4, HVEM, HHLA2, CTLA-4, LAG-3, TIM-3, BTLA, CD160,CEACAM-1, LAIR1, TGFβ, IL-10, Siglec family protein, KIR, CD96, TIGIT,NKG2A, CD112, CD47, SIRPA or CD244.

Exemplary anti-PD-1 antibodies that may be used in the methods of theinvention are those described herein and in U.S. Pat. Nos. 5,897,862 and7,488,802, and in Int. Patent Publ. Nos. WO2004/004771, WO2004/056875,WO2006/121168, WO2008/156712, WO2010/029435, WO2010/036959,WO2011/110604, WO2012/145493, WO2014/194302, WO2014/206107,WO2015/036394, WO2015/035606, WO2015/085847, WO2015/112900 andWO2015/112805. Exemplary anti-PD1 antibodies include KEYTRUDA®(pembrolizumab) and OPDIVO® (nivolumab).

In some embodiments, the antibodies of the invention are administered incombination with a soluble PD-1 ligand.

In some embodiments, the soluble PD-1 ligand is soluble PD-L1 or solublePD-L2 fused to an Fc.

In some embodiments, the soluble PD-1 ligand is AMP-224.

In some embodiments, the antibodies of the invention are administered incombination with an anti-PD-L1 antibody, or antigen-binding fragmentsthereof.

Exemplary PD-L1 antibodies that may be used in the methods of theinvention are antibodies MDPL3280A (Genentech/Roche) and other humanmonoclonal antibodies disclosed in U.S. Pat. No. 7,943,743 and U.S.Patent Publ. No. 20120039906. Other anti-PD-L1 binding agents includeYW243.55.S70 (heavy and light chain variable regions are shown in SEQ IDNOs 20 and 21 in WO2010/077634) and MDX-1105 (also referred to asBMS-936559, and, e.g., anti-PD-L1 binding agents disclosed inWO2007/005874). The VH and the VL sequences of anti-PD-L1 antibodiesdurvalumab, atezolimumab and avelumab that may be used are disclosedherein.

Exemplary PD-L2 antibodies that may be used in the methods of theinvention are those described in U.S. Pat. Nos. 8,080,636, 8,188,238,U.S. Patent Publ. No. 20110271358 and Int. Patent Publ. No.WO2012145493.

Exemplary B7-H4 antibodies that may be used in the methods of theinvention are those described in U.S. Pat. Nos. 7,888,477, 8,609,816,7,931,896, European Patent No. 1817055, U.S. Patent Publ. No.US20140037551 and US2014029486, and Int. Patent Publ. Nos. WO2014/100483and WO2014/159835.

Exemplary anti-CTLA-4 antibodies that may be used in the methods of theinvention are ipilimumab (MDX-010, CAS No. 477202-00-9) and tremelimumab(IgG2 monoclonal antibody available from Pfizer, formerly known asticilimumab, CP-675,206).

Exemplary anti-LAG-3 antibodies that may be used in the methods of theinvention are those described for example in Int. Patent Publ. Nos.WO2008/132601 and WO2010/019570.

Exemplary anti-CEACAM-1 antibodies that may be used in the methods ofthe invention are those described in U.S. Pat. No. 8,598,322 and in U.S.Patent Publ. Nos. US2004/0047858, US20140271618 and US20120100158.Without wishing to be bound by any particular theory, CEACAM-1 has beendescribed as a ligand and partner of TIM-3 (see e.g., Int. Patent Publ.No. WO2014/022332). Synergistic in vivo effect of the combination ofanti-TIM-3 and anti-CEACAM-1 antibodies have been detected in xenograftcancer models (see e.g., Int. Patent Publ. No. WO2014/022332). Tumorsmay use CEACAM-1 to inhibit the immune system. Therefore, anti-CEACAM-1antibodies may be used in combination with the antibodies of theinvention described herein.

Exemplary anti-LAIR1 antibodies that may be used in the methods of theinvention are those described in U.S. Pat. No. 6,479,638 and Int. PatentPubl. No. WO2010/078580.

Exemplary anti-CD96 antibodies that may be used in the methods of theinvention are those described in Int. Patent Publ. No. WO2015/024060.

Exemplary anti-TIM-3 antibodies that may be used in the methods of theinvention are those described herein and in Int. Patent Publ. Nos.WO2011/155607, WO2013/006490 and WO2015/117002.

Exemplary anti-TIGIT antibodies that may be used in the methods of theinvention are those described in U.S. Patent Publ. Nos. US20140056890and US20150216970. An exemplary anti-TIGIT antibody is RG-6058(MTIG-7192A).

TIGIT expression was found herein to be elevated in CD8⁺ T cellsisolated from tumors after anti-TIM-3 antibody treatment in animalmodels of cancer. Therefore, therapeutic administration of antagonisticantibodies specifically binding TIGIT to a subject who has alreadyreceived or is receiving anti-TIM-3 antibody therapy, is refractory tothe anti-TIM-3 antibody treatment or has relapsed after or during theanti-TIM-3 antibody treatment may improve the clinical outcome of thepatients.

The invention also provides a method of treating a cancer in a subject,comprising administering to the subject in need thereof atherapeutically effective amount of an antagonistic antibody thatspecifically binds TIM-3 and an antagonistic antibody that specificallybinds TIGIT for a time sufficient to treat the cancer.

In some embodiments, the antagonistic antibody that specifically bindsTIGIT is administered after administration of the antagonistic antibodyspecifically binding TIM-3.

In some embodiments, the antagonistic antibody that specifically bindsTIGIT and the antagonistic antibody specifically binding TIM-3 areadministered concurrently as single agents or sequentially as singleagents in any order.

Exemplary anti-BTLA antibodies that may be used in the methods of theinvention are those described in U.S. Pat. Nos. 8,546,541, 7,479,544,8,188,232, 8,247,537, 8,563,694 and in Int. Patent Publ. No.WO2014184360.

Exemplary anti-HVEM antibodies that may be used in the methods of theinvention are those described in U.S. Patent Publ. No. US20110280866.

Exemplary CD47 antibodies that may be used in the methods of theinvention are those described in U.S. Pat. No. 8,101,719.

Exemplary CD244 antibodies that may be used in the methods of theinvention include those described in U.S. Pat. No. 5,688,690.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-TIM-3 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-PD-L1 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-PD-L2 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-VISTA antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-BTNL2 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-B7-H3 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-B7-H4 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-HVEM antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-HLA2 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CTLA-4 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-LAG-3 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-TIM-3 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-BTLA antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CD160 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CEACAM-1 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-LAIR1 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-TGFβ antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-IL-10 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-TIGIT antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-KIR antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-NKG2A antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CD112 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CD47 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-SIRPA antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CD244antibody or antigen-binding fragment thereof.

The immune inhibitory molecules may regulate or synergistically regulateT-cell functions to promote tumoral immune escape. Therefore,combination therapies with two or more inhibitors of the inhibitorymolecules may provide an improved therapy to a patient when compared tomonotherapy alone.

In some embodiments, the antibodies of the invention are administered incombination with an activator of an activating molecule.

In some embodiments, the antibodies of the invention are administered incombination with an activator of an activating molecule CD86, CD80,CD28, ICOS, ICOS ligand, TMIGD2, CD40, GITR ligand, 4-1BB ligand, OX40ligand, CD70, CD40L, TNFRSF25, LIGHT, GITR, OX-40, CD27, CD137, NKG2D,CD48, CD226 or MICA.

Activation of activating molecules may be performed using for examplesoluble ligands or ligand derivatives of the activating molecules,peptides or agonistic antibodies.

In some embodiments, the activator of the activating molecule is asoluble ligand of the T cell activating molecule.

In some embodiments, the activator of the activating molecule is anagonistic antibody specifically binding the activating molecule.

Exemplary anti-CD40 antibodies that may be used in the methods of theinvention include CP-870,893 and humanized S2C6 described in U.S. Pat.No. 7,288,251 (antibody 21.4.1) and U.S. Pat. No. 8,303,955,respectively, and anti-CD40 antibodies described in Int. Patent Publ.Nos. WO2001/056603, WO2001/083755, WO2013/034904 and WO2014/070934.

Exemplary GITR agonists include, e.g., GITR fusion proteins andanti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as, aGITR fusion protein described in U.S. Pat. No. 6,111,090, EuropeanPatent No. 090505B1, U.S. Pat. No. 8,586,023, Int. Patent. Publ. Nos.WO2010/003118 and WO2011/090754, or an anti-GITR antibody described inU.S. Pat. Nos. 7,025,962, 7,812,135, 8,388,967, 8,591,886 and 7,618,632,European Patent Nos. 1947183 and 1866339, or Int. Patent Publ. Nos.WO2011/028683, WO2013/039954, WO2005/007190, WO2007/133822,WO2005/055808, WO1999/40196, WO2001/03720, WO1999/20758, WO2006/083289,WO2005/115451 and WO2011/051726.

GITR expression was found herein to be elevated in CD8⁺ T cells isolatedfrom tumors after anti-PD-1 antibody treatment in animal models ofcancer. The restoration of GITR expression on TILs by anti-PD-1treatment supports that combination therapy with anti-GITR and anti-PD-1antibodies may improve the clinical outcome of the patients.

The invention also provides a method of treating a cancer in a subject,comprising administering to the subject in need thereof atherapeutically effective amount of an antagonistic antibody thatspecifically binds PD-1 and an agonistic antibody that specificallybinds GITR for a time sufficient to treat the cancer.

In some embodiments, the agonistic antibody that specifically binds GITRis administered after administration of the antagonistic antibodyspecifically binding PD-1.

In some embodiments, the agonistic antibody that specifically binds GITRand the antagonistic antibody specifically binding PD-1 are administeredconcurrently as single agents or sequentially as single agents in anyorder.

Exemplary OX40 antibodies that may be used in the methods of theinvention include those described in U.S. Pat. Nos. 8,133,983,7,960,515, U.S. Patent Publ. No. 20130280275 and Int. Patent Publ. Nos.WO2013028231 and WO2014148895.

An exemplary OX40 antibody that may be used in the methods of theinvention is an antibody comprising the VH of SEQ ID NO: 309 and the VLof SEQ ID NO: 310.

Another exemplary OX40 antibody that may be used in the methods of theinvention is an antibody comprising the VH of SEQ ID NO: 311 and the VLof SEQ ID NO: 312.

OX40 expression was found herein to be elevated in CD8⁺ T cells isolatedfrom tumors after anti-PD-1 antibody treatment in animal models ofcancer. The restoration of OX40 expression on TILs by anti-PD-1treatment supports that combination therapy with anti-OX40 and anti-PD-1antibodies may improve the clinical outcome of the patients.

The invention also provides a method of treating a cancer in a subject,comprising administering to the subject in need thereof atherapeutically effective amount of an antagonistic antibody thatspecifically binds PD-1 and an agonistic antibody that specificallybinds OX40 for a time sufficient to treat the cancer.

In some embodiments, the agonistic antibody that specifically binds OX40is administered after administration of the antagonistic antibodyspecifically binding PD-1.

In some embodiments, the agonistic antibody that specifically binds OX40and the antagonistic antibody specifically binding PD-1 are administeredconcurrently as single agents or sequentially as single agents in anyorder.

Exemplary CD70 antibodies that may be used in the methods of theinvention include those described in U.S. Patent Publ. No.US20130336976.

Exemplary TNFRSF25 antibodies that may be used in the methods of theinvention include those described in U.S. Pat. No. 7,708,996.

Exemplary CD27 antibodies that may be used in the methods of theinvention include those described in U.S. Patent Publ. No.US20130336976.

Exemplary CD137 antibodies that may be used in the methods of theinvention include those described in U.S. Pat. Nos. 6,974,863,6,303,121, 7,138,500, 7,288,638, 8,716,452, 8,821,867 and in U.S. PatentPubl. No. US20130149301.

CD137 expression was found herein to be elevated in CD8⁺ T cellsisolated from tumors after anti-PD-1 antibody treatment in animal modelsof cancer. The restoration of CD137 expression on TILs by anti-PD-1treatment supports that combination therapy with anti-CD137 andanti-PD-1 antibodies may improve the clinical outcome of the patients.

The invention also provides a method of treating a cancer in a subject,comprising administering to the subject in need thereof atherapeutically effective amount of an antagonistic antibody thatspecifically binds PD-1 and an agonistic antibody that specificallybinds CD137 for a time sufficient to treat the cancer.

In some embodiments, the agonistic antibody that specifically bindsCD137 is administered after administration of the antagonistic antibodyspecifically binding PD-1.

In some embodiments, the agonistic antibody that specifically bindsCD137 and the antagonistic antibody specifically binding PD-1 areadministered concurrently as single agents or sequentially as singleagents in any order.

Exemplary NKG2D antibodies that may be used in the methods of theinvention include those described in U.S. Patent Publ. No.US20110150870.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CD86 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CD80 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CD28 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-ICOS antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-ICOS ligand antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-TMIGD2 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CD40 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-GITR ligand antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-4-1BB ligand antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-OX40 ligand antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CD70 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CD40L antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-TNFRSF25 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-LIGHT antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-GITR antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-OX40 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CD27 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CD137 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-NKG2D antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CD48 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-CD226 antibody or antigen-binding fragment thereof.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with ananti-MICA antibody or antigen-binding fragment thereof.

The combination of antibodies recited herein can be administeredseparately, e.g., as separate antibodies, or linked, e.g., as abispecific or trispecific antibody molecule.

The efficacy of the combinations described herein may be tested inanimal models known in the art.

Antibodies of the invention described herein may be administered incombination with a vaccine.

Exemplary vaccines are immunogenic agents, such as cancerous cells,purified tumor antigens (including recombinant proteins, antigenepitopes, peptides and carbohydrate molecules), tumor antigens deliveredto a patient via gene therapy, cells, and cells transfected with genesencoding immune stimulating cytokines. Exemplary vaccines that may beused include peptides of melanoma antigens, such as peptides of gp100,MAGE antigens, Trp-2, MARTI and/or tyrosinase, or tumor cellstransfected to express the cytokine GM-CSF, DNA-based vaccines,RNA-based vaccines, and viral transduction-based vaccines, peptides orprostate antigens or peptides of lung cancer antigens. The cancervaccine may be prophylactic or therapeutic.

Many experimental strategies for vaccination against tumors have beendevised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCOEducational Book Spring: 60-62; Logothetis, C., 2000, ASCO EducationalBook Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring:414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see alsoRestifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 inDeVita, V. et al. (eds.), 1997, Cancer: Principles and Practice ofOncology. Fifth Edition). In one of these strategies, a vaccine isprepared using autologous or allogeneic tumor cells. These cellularvaccines have been shown to be most effective when the tumor cells aretransduced to express GM-CSF. GM-CSF has been shown to be a potentactivator of antigen presentation for tumor vaccination (Dranoff et al.,(1993) Proc Natl Acad Sci U.S.A. 90: 3539-43).

The antibodies of the invention described herein may be administered incombination with one or a collection of recombinant proteins and/orpeptides expressed in or on a tumor in order to generate an immuneresponse to these proteins. These proteins are normally viewed by theimmune system as self-antigens and are therefore tolerant to them. Thetumor antigen may also include the protein telomerase, which is requiredfor the synthesis of telomeres of chromosomes and which is expressed inmore than 85% of human cancers and in only a limited number of somatictissues (Kim et al., (1994) Science 266: 2011-2013). Tumor antigens mayalso be “neo-antigens” expressed in or on cancer cells as a result ofsomatic mutations that alter protein sequence or create fusion proteinsbetween two unrelated sequences (e.g., bcr-abl in the Philadelphiachromosome), or idiotype from B cell tumors. The tumor antigens may beantigen epitopes of prostate specific antigen (PSA), mesothelin,prostate-specific membrane antigen (PSMA), synovial sarcoma X2 (SSX2),NKX3.1, prostatic acidic phosphatase (PAP), or epidermal growth factorreceptors, or peptides specific for variants of EGFR such as thewell-known EGFRvIII overexpressed on tumor cells.

Other tumor vaccines may include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV), and Epstein-Barrvirus (EBV). Another form of tumor specific antigens which may be usedin combination with the antibodies of the invention described herein ispurified heat shock proteins (HSP) isolated from the tumor tissueitself. HSP contain fragments of proteins from the tumor cells and arehighly efficient at delivery to antigen presenting cells for elicitingtumor immunity (Suot and Srivastava (1995) Science 269:1585-1588; Tamuraet al., (1997) Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that may beused to prime antigen-specific responses. DC's may be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al., (1998) Nature Medicine 4: 328-332). DCsmay also be transduced by genetic means to express these tumor antigens.DCs have also been fused directly to tumor cells for the purposes ofimmunization (Kugler et al., (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization may be effectively combined withthe antibodies of the invention described herein to activate more potentanti-tumor responses.

In some embodiments, the vaccine is a polypeptide or a fragment thereof,or a DNA or a RNA encoding the polypeptide or fragment thereof expressedon tumor cells.

In some embodiments, the polypeptide or fragment thereof expressed ontumor cells is PSMA.

In some embodiments, the polypeptide or fragment thereof expressed ontumor cells is mesothelin.

In some embodiments, the polypeptide or fragment thereof expressed ontumor cells is EGFR or EGFR variant such as EGFRvIII.

In some embodiments, the polypeptide or fragment thereof expressed ontumor cells is PAP.

In some embodiments, the polypeptide or fragment thereof expressed ontumor cells is synovial sarcoma X2 (SSX2).

In some embodiments, the polypeptide or fragment thereof expressed ontumor cells is NKX3.1.

In some embodiments, the tumor cells are melanoma, lung cancer, squamousnon-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectalcancer, prostate cancer, castration-resistant prostate cancer, ovariancancer, gastric cancer, liver cancer, pancreatic cancer, thyroid cancer,squamous cell carcinoma of the head and neck, carcinomas of theesophagus or gastrointestinal tract or breast cancer cells.

In some embodiments, the antibodies of the invention are administered incombination with a renal carcinoma (RCC) vaccine.

In some embodiments, the antibodies of the invention are administered incombination with a lung cancer vaccine.

In some embodiments, the antibodies of the invention are administered incombination with a prostate cancer vaccine.

In some embodiments, the antibodies of the invention are administered incombination with a lung cancer vaccine.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with a tumorvaccine comprising a peptide fragment of EGFR or EGFRvIII, or a vectorencoding the peptide fragment of EGFR or EGFRvIII.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with a tumorvaccine comprising a peptide fragment of mesothelin, or a vectorencoding the peptide fragment of mesothelin.

In some embodiments, the antagonistic antibodies specifically bindingPD-1 of the invention, the antagonistic antibodies specifically bindingTIM-3 or the invention, or the antagonistic bispecific PD-1/TIM-3antibodies of the invention are administered in combination with a tumorvaccine comprising a peptide fragment of prostate specific antigen, or avector encoding the peptide fragment of prostate specific antigen.

Suitable vectors that may be used in the methods of the invention arewell known and include lentiviral vectors, adenoviral vectors, minimalnucleic acid vector (MNAV), vaccinia virus, flow pox virus, Alphavirus-derived VRP, Saccharomyces cerevisiae, MVA, Listeriamoonocytogenes, pVAX-based plasmid, see e.g. Pol et al., (2014)Oncoimmunology 1(3):e28185.

The antibodies of the invention may be administered in combination witha standard of care cancer treatment.

The antibodies of the invention described herein may be administered incombination with a standard of care cancer chemotherapeutic regimes. Inthese instances, it may be possible to reduce the dose ofchemotherapeutic reagent administered (Mokyr et al., (1998) CancerResearch 58: 5301-5304).

In some embodiments, the antibodies of the invention may be administeredin combination with one or more of other antibody molecules,chemotherapy, other anti-cancer therapy (e.g., targeted anti-cancertherapies, or oncolytic drugs), cytotoxic agents, cytokines, surgicaland/or radiation procedures.

Exemplary cytotoxic agents that may be administered in combination withthe antibodies of the invention include antimicrotubule agents,topoisomerase inhibitors, anti-metabolites, mitotic inhibitors,alkylating agents, anthracyclines, vinca alkaloids, intercalatingagents, agents capable of interfering with a signal transductionpathway, agents that promote apoptosis, proteosome inhibitors, andradiation (e.g., local or whole body irradiation).

Standard of care therapeutics include anastrozole (Arimidex®),bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan(Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®),N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®),carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin(Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®),etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil(Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine(difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®),ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®),leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine(Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®),paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin,polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate(Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine(Tirazone®), topotecan hydrochloride for injection (Hycamptin®),vinblastine (Velban®), vincristine (Oncovin®), vinorelbine (Navelbine®),Ibrutinib, idelalisib, and brentuximab vedotin.

Exemplary alkylating agents include, nitrogen mustards, ethyleniminederivatives, alkyl sulfonates, nitrosoureas and triazenes: uracilmustard (Aminouracil Mustard®, Chlorethaminaci®, Demethyldopan®,Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil Nitrogen Mustard®,Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine(Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®,Procytox®, Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®),chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®),triethylenemelamine (Hemel®, Hexalen®, Hexastat®),triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa(Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®),lomustine (CeeNU®) and streptozocin (Zanosar®). Additional exemplaryalkylating agents include, oxaliplatin (Eloxatin®), temozolomide(Temodar® and Temodal®), dactinomycin (also known as actinomycin-D,Cosmegen®), altretamine (also known as hexamethylmelamine (HMM),Hexalen®), bendamustine (Treanda®), carboplatin (Paraplatin®), lomustine(also known as CCNU, CeeNU®), cisplatin (also known as CDDP, Platinol®and Platinol®-AQ), chlorambucil (Leukeran®), prednumustine, procarbazine(Matulane®), and thiotepa (also known as thiophosphoamide, TESPA andTSPA, Thioplex®).

Exemplary anthracyclines include, e.g., doxorubicin (Adriamycin® andRubex®); bleomycin (Lenoxane®), daunorubicin (dauorubicin hydrochloride,daunomycin, and rubidomycin hydrochloride, Cerubidine®), daunorubicinliposomal (daunorubicin citrate liposome, DaunoXome®), mitoxantrone(DHAD, Novantrone®), epirubicin (Ellence™), idarubicin (Idamycin®,Idamycin PFS®), mitomycin C (Mutamycin®), geldanamycin, herbimycin,ravidomycin, and desacetylravidomycin.

Exemplary vinca alkaloids that may be used in combination with theantibodies of the invention include vinorelbine tartrate (Navelbine®),vincristine (Oncovin®), and vindesine (Eldisine®), vinblastine (alsoknown as vinblastine sulfate, vincaleukoblastine and VLB, Alkaban-AQ®and Velban®) and vinorelbine (Navelbine®).

Exemplary proteosome inhibitors that may be used in combination with theantibodies of the invention are bortezomib (Velcade®); carfilzomib(Kyprolis®), ixazomib (Ninlaro®), marizomib (NPI-0052) and delanzomib(CEP-18770).

In some embodiments, the antibodies of the invention are administered incombination with a tyrosine kinase inhibitor (e.g., a receptor tyrosinekinase (RTK) inhibitor). Exemplary tyrosine kinase inhibitor include anepidermal growth factor (EGF) pathway inhibitor (e.g., an epidermalgrowth factor receptor (EGFR) inhibitor), a vascular endothelial growthfactor (VEGF) pathway inhibitor (e.g., a vascular endothelial growthfactor receptor (VEGFR) inhibitor (e.g., a VEGFR-1 inhibitor, a VEGFR-2inhibitor, a VEGFR-3 inhibitor), a platelet derived growth factor (PDGF)pathway inhibitor (e.g., a platelet derived growth factor receptor(PDGFR) inhibitor (e.g., a PDGFR-β inhibitor), a RAF-1 inhibitor, a KITinhibitor and a RET inhibitor. In some embodiments, the secondtherapeutic is axitinib (AG013736), bosutinib (SKI-606), cediranib(RECENTIN™, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib(TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B,STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701),neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib,SU5416), sunitinib (SUTENT®, SU11248), toceranib (PALLADIA®), vandetanib(ZACTIMA®, ZD6474), vatalanib (PTK787, PTK/ZK), trastuzumab(HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®), cetuximab(ERBITUX®), panitumumab (VECTIBIX®), ranibizumab (Lucentis®), nilotinib(TASIGNA®), sorafenib (NEXAVAR®), alemtuzumab (CAMPATH®), gemtuzumabozogamicin (MYLOTARG®), ENMD-2076, PCI-32765, AC220, dovitinib lactate(TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903,PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120(VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154,CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, XL228,AEE788, AG-490, AST-6, BMS-599626, CUDC-101, PD153035, pelitinib(EKB-569), vandetanib (zactima), WZ3146, WZ4002, WZ8040, ABT-869(linifanib), AEE788, AP24534 (ponatinib), AV-951 (tivozanib), axitinib,BAY 73-4506 (regorafenib), brivanib alaninate (BMS-582664), brivanib(BMS-540215), cediranib (AZD2171), CHIR-258 (dovitinib), CP 673451,CYC116, E7080, Ki8751, masitinib (AB1010), MGCD-265, motesanibdiphosphate (AMG-706), MP-470, OSI-930, pazopanib hydrochloride,PD173074, Sorafenib Tosylate (Bay 43-9006), SU 5402, TSU-68 (SU6668),vatalanib, XL880 (GSK1363089, EXEL-2880). Selected tyrosine kinaseinhibitors are chosen from sunitinib, erlotinib, gefitinib, orsorafenib. In some embodiments, the EGFR inhibitor is a bispecificEGFRc-Met antibody (EM-1 mAb) comprising the heavy and the light chainsof SEQ ID NOs: 249, 250, 251 and 252 (US2014/0141000).

In some embodiments, the antibodies of the invention are administered incombination with Vascular Endothelial Growth Factor (VEGF) receptorinhibitors, including bevacizumab (Avastin®), axitinib (Inlyta®),brivanib alaninate (BMS-582664,(S)—((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate),sorafenib (Nexavar®); Pazopanib (Votrient®), sunitinib malate (Sutent®),cediranib (AZD2171, CAS 288383-20-1), vargatef (BIBF1120, CAS928326-83-4), foretinib (GSK1363089), telatinib (BAY57-9352, CAS332012-40-5), apatinib (YN968D1, CAS 811803-05-1), imatinib (Gleevec®),ponatinib (AP24534, CAS 943319-70-8), tivozanib (AV951, CAS475108-18-0), regorafenib (BAY73-4506, CAS 755037-03-7), vatalanibdihydrochloride (PTK787, CAS 212141-51-0), brivanib (BMS-540215, CAS649735-46-6), vandetanib (Caprelsa® or AZD6474), motesanib diphosphate(AMG706, CAS 857876-30-3,N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyflamino]-3-pyridinecarboxamide,described in PCT Publication No. WO 02/066470), dovitinib dilactic acid(TKI258, CAS 852433-84-2), linfanib (ABT869, CAS 796967-16-3);Cabozantinib (XL184, CAS 849217-68-1), lestaurtinib (CAS 111358-88-4);N-[5-[[[5-(1,1-Dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide(BMS38703, CAS 345627-80-7);(3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol(BMS690514);N-(3,4-Dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)—octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine(XL647, CAS 781613-23-8);4-Methyl-3-[[1-methyl-6-(3-pyridinyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]amino]-N-[3-(trifluoromethyl)phenyl]-benzamide(BHG712, CAS 940310-85-0); and aflibercept (Eylea®).

In some embodiments, the antibodies of the invention are administered incombination with a PI3K inhibitor. In one embodiment, the PI3K inhibitoris an inhibitor of delta and gamma isoforms of PI3K. Exemplary PI3Kinhibitors that may be used are described in, e.g., WO 2010/036380, WO2010/006086, WO 09/114870, WO 05/113556, GSK 2126458, GDC-0980,GDC-0941, Sanofi XL147, XL756, XL147, PF-46915032, BKM 120, CAL-101, CAL263, SF1126, PX-886, and a dual PI3K inhibitor (e.g., Novartis BEZ235).

In some embodiments, the antibodies of the invention are administered incombination with a mTOR inhibitor, e.g., one or more mTOR inhibitorschosen from one or more of rapamycin, temsirolimus (TORISEL®), AZD8055,BEZ235, BGT226, XL765, PF-4691502, GDC0980, SF1126, OSI-027, GSK1059615,KU-0063794, WYE-354, Palomid 529 (P529), PF-04691502, or PKI-587.ridaforolimus (formally known as deferolimus, (1R,2R,4S)-4-[(2R)—2[(1R,95,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described inPCT Publication No. WO 03/064383); everolimus (Afinitor® or RAD001);rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3);emsirolimus,(5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenypmethanol(AZD8055);2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502, CAS 1013101-36-4); andN2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-(SEQ ID NO: 237), inner salt (SF1126, CAS 936487-67-1), and XL765.

In some embodiments, the antibodies of the invention are administered incombination with a BRAF inhibitor, e.g., GSK2118436, RG7204, PLX4032,GDC-0879, PLX4720, and sorafenib tosylate (Bay 43-9006).

In some embodiments, the antibodies of the invention are administered incombination with a MEK inhibitor.

In some embodiments, the antibodies of the invention are administered incombination with a JAK2 inhibitor, e.g., CEP-701, INCB18424, CP-690550(tasocitinib).

In some embodiments, the antibodies of the invention are administered incombination with paclitaxel or a paclitaxel agent, e.g., TAXOL®,protein-bound paclitaxel (e.g., ABRAXANE®). Exemplary paclitaxel agentsinclude nanoparticle albumin-bound paclitaxel (ABRAXANE, marketed byAbraxis Bioscience), docosahexaenoic acid bound-paclitaxel(DHA-paclitaxel, Taxoprexin, marketed by Protarga), polyglutamatebound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX,marketed by Cell Therapeutic), the tumor-activated prodrug (TAP), ANG105(Angiopep-2 bound to three molecules of paclitaxel, marketed byImmunoGen), paclitaxel-EC-1 (paclitaxel bound to the erbB2-recognizingpeptide EC-1; see Li et al., Biopolymers (2007) 87:225-230), andglucose-conjugated paclitaxel (e.g., T-paclitaxel methyl2-glucopyranosyl succinate, see Liu et al., (2007) Bioorganic &Medicinal Chemistry Letters 17:617-620).

In some embodiments, the antibodies of the invention are administered incombination with a cellular immunotherapy (e.g., Provenge (e.g.,Sipuleucel)), and optionally in combination with cyclophosphamide.

Exemplary therapeutic agents that may be used in combination with theantibodies of the invention for treatment of pancreatic cancer include achemotherapeutic agent, e.g., paclitaxel or a paclitaxel agent (e.g., apaclitaxel formulation such as TAXOL, an albumin-stabilized nanoparticlepaclitaxel formulation (e.g., ABRAXANE) or a liposomal paclitaxelformulation); gemcitabine (e.g., gemcitabine alone or in combinationwith AXP107-11); other chemotherapeutic agents such as oxaliplatin,5-fluorouracil, capecitabine, rubitecan, epirubicin hydrochloride,NC-6004, cisplatin, docetaxel (e.g., TAXOTERE), mitomycin C, ifosfamide;interferon; tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g.,erlotinib, panitumumab, cetuximab, nimotuzumab); HER2/neu receptorinhibitor (e.g., trastuzumab); dual kinase inhibitor (e.g., bosutinib,saracatinib, lapatinib, vandetanib); multikinase inhibitor (e.g.,sorafenib, sunitinib, XL184, pazopanib); VEGF inhibitor (e.g.,bevacizumab, AV-951, brivanib); radioimmunotherapy (e.g., XR303); cancervaccine (e.g., GVAX, survivin peptide); COX-2 inhibitor (e.g.,celecoxib); IGF-1 receptor inhibitor (e.g., AMG 479, MK-0646); mTORinhibitor (e.g., everolimus, temsirolimus), IL-6 inhibitor (e.g., CNTO328); cyclin-dependent kinase inhibitor (e.g., P276-00, UCN-01); AlteredEnergy Metabolism-Directed (AEMD) compound (e.g., CPI-613); HDACinhibitor (e.g., vorinostat); TRAIL receptor 2 (TR-2) agonist (e.g.,conatumumab); MEK inhibitor (e.g., AS703026, selumetinib, GSK1120212);Raf/MEK dual kinase inhibitor (e.g., RO5126766), Notch signalinginhibitor (e.g., MK0752), monoclonal antibody-antibody fusion protein(e.g., L19IL2), curcumin; HSP90 inhibitor (e.g., tanespimycin,STA-9090), rIL-2; denileukin diftitox; topoisomerase 1 inhibitor (e.g.,irinotecan, PEP02); statin (e.g., simvastatin), Factor VIM inhibitor(e.g., PCI-27483), AKT inhibitor (e.g., RX-0201), hypoxia-activatedprodrug (e.g., TH-302), metformin hydrochloride, gamma-secretaseinhibitor (e.g., RO4929097), ribonucleotide reductase inhibitor (e.g.,3-AP), immunotoxin (e.g., HuC242-DM4), PARP inhibitor (e.g., KU-0059436,veliparib), CTLA-4 inhibitor (e.g., CP-675,206, ipilimumab), AdV-tktherapy, proteasome inhibitor (e.g., bortezomib (Velcade), NPI-0052),thiazolidinedione (e.g., pioglitazone), NPC-1C; Aurora kinase inhibitor(e.g., R763/AS703569), CTGF inhibitor (e.g., FG-3019), siG12D LODER andradiation therapy (e.g., tomotherapy, stereotactic radiation, protontherapy), surgery, and a combination thereof. In certain embodiments, acombination of paclitaxel or a paclitaxel agent, and gemcitabine can beused with the antibodies of the invention.

Exemplary therapeutic agents that may be used in combination with theantibodies of the invention for treatment of small cell lung cancer(SCLC) include approved drugs for treatment of SCLC such as methotrexate(Folex®, Mexate®), everolimus (Afinitor®), doxorubicin hydrochloride,etoposide phosphate (Etopophos®), topotecan hydrochloride (Hycamtin®),mechlorethamine hydrochloride (Mustargen®), topotecan hydrochloride.Other therapeutic agents that may be used are carboplatin, cisplatin,oxaliplatin, irinotecan, gemcitabine, liposomal SN-38, bendamustine,temozolomide, belotecan, NK012, FR901228, flavopiridol), tyrosine kinaseinhibitor (e.g., EGFR inhibitor (e.g., erlotinib, gefitinib, cetuximab,panitumumab), multikinase inhibitor (e.g., sorafenib, sunitinib), VEGFinhibitor (e.g., bevacizumab, vandetanib), cancer vaccine (e.g., GVAX);Bcl-2 inhibitor (e.g., oblimersen sodium, ABT-263), proteasome inhibitor(e.g., bortezomib (Velcade), NPI-0052), paclitaxel or a paclitaxelagent; docetaxel, IGF-1 receptor inhibitor (e.g., AMG 479), HGF/SFinhibitor (e.g., AMG 102, MK-0646), chloroquine, Aurora kinase inhibitor(e.g., MLN8237), radioimmunotherapy (e.g., TF2), HSP90 inhibitor (e.g.,tanespimycin, STA-9090), mTOR inhibitor (e.g., everolimus),Ep-CAM/CD3-bispecific antibody (e.g., MT110), CK-2 inhibitor (e.g.,CX-4945), HDAC inhibitor (e.g., belinostat), SMO antagonist (e.g., BMS833923), peptide cancer vaccine, and radiation therapy (e.g.,intensity-modulated radiation therapy (IMRT), hypofractionatedradiotherapy, hypoxia-guided radiotherapy), surgery, and combinationsthereof.

Exemplary therapeutic agents that may be used in combination with theantibodies of the invention for treatment of non-small cell lung cancerinclude approved drugs for treatment of NSCLC including methotrexate(Folex®, Mexate®), paclitaxel (Abraxane®), afatinib (Gilotrif®),everolimus (Afinitor®), alectinib (Alecensa®), pemetrexed disodium(Alimta®), bevacizumab (Avastin®), carboplatin, ceritinib (Zykadia®),crizotinib (Xalkori®), ramucirumab (Cyramza®), docetaxel, everolimus(Afinitor®), gefitinib (Iressa®), afatinib dimaleate (Gilotrif®),gemcitabine hydrochloride (Gmezar®), pembrolizumab (Keytruda®),mechlorethamine hydrochloride (Mustargen®), vinorelbine tartrate(Navelbine®), necitumumab (Portrazza®), nivolumab (Opdivo®),osimertinib, paclitaxel (Taxol®), carboplatin, pemetrexed disodium,ramucirumab (Cyramza®), osimertinib (Tagrisso®). Other therapeuticagents that may be used are vinorelbine, cisplatin, docetaxel,pemetrexed disodium, etoposide, gemcitabine, carboplatin, liposomalSN-38, TLK286, temozolomide, topotecan, pemetrexed disodium,azacitidine, irinotecan, tegafur-gimeracil-oteracil potassium,sapacitabine), tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g.,erlotinib, gefitinib, cetuximab, panitumumab, necitumumab, PF-00299804,nimotuzumab, RO5083945), MET inhibitor (e.g., PF-02341066, ARQ 197),PI3K kinase inhibitor (e.g., XL147, GDC-0941), Raf/MEK dual kinaseinhibitor (e.g., RO5126766), PI3K/mTOR dual kinase inhibitor (e.g.,XL765), SRC inhibitor (e.g., dasatinib), dual inhibitor (e.g., BIBW2992, GSK1363089, ZD6474, AZD0530, AG-013736, lapatinib, MEHD7945A,linifanib), multikinase inhibitor (e.g., sorafenib, sunitinib,pazopanib, AMG 706, XL184, MGCD265, BMS-690514, R935788), VEGF inhibitor(e.g., endostar, endostatin, bevacizumab, cediranib, BIBF 1120,axitinib, tivozanib, AZD2171), cancer vaccine (e.g., BLP25 liposomevaccine, GVAX, recombinant DNA and adenovirus expressing L523S protein),Bcl-2 inhibitor (e.g., oblimersen sodium), proteasome inhibitor (e.g.,bortezomib, carfilzomib, NPI-0052, MLN9708), paclitaxel or a paclitaxelagent, docetaxel, IGF-1 receptor inhibitor (e.g., cixutumumab, MK-0646,OSI 906, CP-751,871, BIIB022), hydroxychloroquine, HSP90 inhibitor(e.g., tanespimycin, STA-9090, AUY922, XL888), mTOR inhibitor (e.g.,everolimus, temsirolimus, ridaforolimus), Ep-CAM/CD3-bispecific antibody(e.g., MT110), CK-2 inhibitor (e.g., CX-4945), HDAC inhibitor (e.g., MS275, LBH589, vorinostat, valproic acid, FR901228), DHFR inhibitor (e.g.,pralatrexate), retinoid (e.g., bexarotene, tretinoin), antibody-drugconjugate (e.g., SGN-15), bisphosphonate (e.g., zoledronic acid), cancervaccine (e.g., belagenpumatucel-L), low molecular weight heparin (LMWH)(e.g., tinzaparin, enoxaparin), GSK1572932A, melatonin, talactoferrin,dimesna, topoisomerase inhibitor (e.g., amrubicin, etoposide,karenitecin), nelfinavir, cilengitide, ErbB3 inhibitor (e.g., MM-121,U3-1287), survivin inhibitor (e.g., YM155, LY2181308), eribulinmesylate, COX-2 inhibitor (e.g., celecoxib), pegfilgrastim, Polo-likekinase 1 inhibitor (e.g., BI 6727), TRAIL receptor 2 (TR-2) agonist(e.g., CS-1008), CNGRC peptide (SEQ ID NO: 225)-TNF alpha conjugate,dichloroacetate (DCA), HGF inhibitor (e.g., SCH 900105), SAR240550,PPAR-gamma agonist (e.g., CS-7017), gamma-secretase inhibitor (e.g.,RO4929097), epigenetic therapy (e.g., 5-azacitidine), nitroglycerin, MEKinhibitor (e.g., AZD6244), cyclin-dependent kinase inhibitor (e.g.,UCN-01), cholesterol-Fus1, antitubulin agent (e.g., E7389),farnesyl-OH-transferase inhibitor (e.g., lonafarnib), immunotoxin (e.g.,BB-10901, SS1 (dsFv) PE38), fondaparinux, vascular-disrupting agent(e.g., AVE8062), PD-L1 inhibitor (e.g., MDX-1105, MDX-1106),beta-glucan, NGR-hTNF, EMD 521873, MEK inhibitor (e.g., GSK1120212),epothilone analog (e.g., ixabepilone), kinesin-spindle inhibitor (e.g.,4SC-205), telomere targeting agent (e.g., KML-001), P70 pathwayinhibitor (e.g., LY2584702), AKT inhibitor (e.g., MK-2206), angiogenesisinhibitor (e.g., lenalidomide), Notch signaling inhibitor (e.g.,OMP-21M18), EGFR/c-Met bispecific antibody EM-1 as described inUS2014/0141000A1, radiation therapy, surgery, and combinations thereof.

Exemplary therapeutic agents that may be used in combination with theantibodies of the invention for treatment of ovarian cancer includeapproved drugs for treatment of ovarian cancer, such as melphalan(Alkeran®), bevacizumab (Avastin®), carboplatin, cyclophosphamide(Clafen®, Cytoxan®), clisplatin, doxorubicin hydrochloride, gemcitabinehydrochloride (Gemzar®), topotecan hydrochloride (Hycamtin®), Olaparib(Lynparza®), carboplatin, cisplatin, paclitaxel (Taxol®), thiotepa andtopotecan hydrochloride. Other therapeutic agents that may be used are,ifosfamide, olaparib, oxaliplatin, pemetrexed disodium, SIG-136,etoposide, decitabine; immunotherapy (e.g., APC8024, oregovomab,OPT-821), tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g.,erlotinib), dual inhibitor (e.g., E7080), multikinase inhibitor (e.g.,AZD0530, JI-101, sorafenib, sunitinib, pazopanib), VEGF inhibitor (e.g.,bevacizumab, BIBF 1120, cediranib, AZD2171), PDGFR inhibitor (e.g.,IMC-3G3), paclitaxel, topoisomerase inhibitor (e.g., karenitecin,Irinotecan), HDAC inhibitor (e.g., valproate, vorinostat), folatereceptor inhibitor (e.g., farletuzumab), angiopoietin inhibitor (e.g.,AMG 386), epothilone analog (e.g., ixabepilone), proteasome inhibitor(e.g., carfilzomib), IGF-1 receptor inhibitor (e.g., OSI 906, AMG 479),PARP inhibitor (e.g., veliparib, AG014699, iniparib, MK-4827), Aurorakinase inhibitor (e.g., MLN8237, ENMD-2076), angiogenesis inhibitor(e.g., lenalidomide), DHFR inhibitor (e.g., pralatrexate),radioimmunotherapeutic agent (e.g., Hu3S193), statin (e.g., lovastatin),topoisomerase 1 inhibitor (e.g., NKTR-102), cancer vaccine (e.g., p53synthetic long peptides vaccine, autologous OC-DC vaccine), mTORinhibitor (e.g., temsirolimus, everolimus), BCR/ABL inhibitor (e.g.,imatinib), ET-A receptor antagonist (e.g., ZD4054), TRAIL receptor 2(TR-2) agonist (e.g., CS-1008), HGF/SF inhibitor (e.g., AMG 102),EGEN-001, Polo-like kinase 1 inhibitor (e.g., BI 6727), gamma-secretaseinhibitor (e.g., RO4929097), Wee-1 inhibitor (e.g., MK-1775),antitubulin agent (e.g., vinorelbine, E7389), immunotoxin (e.g.,denileukin diftitox), SB-485232, vascular-disrupting agent (e.g.,AVE8062), integrin inhibitor (e.g., EMD 525797), kinesin-spindleinhibitor (e.g., 4SC-205), revlimid, HER2 inhibitor (e.g., MGAH22),ErrB3 inhibitor (e.g., MM-121), radiation therapy, and combinationsthereof.

Exemplary therapeutic agents that may be used in combination with theantibodies of the invention for treatment of a myeloma include one ormore of chemotherapy or other anti-cancer agents (e.g., thalidomideanalogs, e.g., lenalidomide), HSCT (Cook, (2008) J Manag Care Pharm.14(7 Suppl):19-25), an anti-TIM-3 antibody (Hallett et al, (2011) J ofAmerican Society for Blood and Marrow Transplantation 17(8):1133-145),tumor antigen-pulsed dendritic cells, fusions (e.g., electrofusions) oftumor cells and dendritic cells, or vaccination with immunoglobulinidiotype produced by malignant plasma cells (reviewed in Yi (2009)Cancer J 15(6):502-10).

Exemplary therapeutics agents that may be used in combination with theantibodies of the invention for treatment of a renal cancer, e.g., arenal cell carcinoma (RCC) or metastatic RCC include drugs approved fortreatment of RCC, including everolimus (Afinitor®), aldesleukin,bevacizumab (Avastin®), axitinib (Inlyta®), cabozantinib-S-Malate(Cabometyx®), aldesleukin (Proleukin®), lenvatinib mesylate (Lenvima®),sorafenib tosylate (Nexavar®), nivolumab (Opdivo®), pazopanibhydrochloride, sorafenib tosylate, sunitinib (Sutent®), temsirolimus(Torisel®) and pazopanib hydrochloride (Votrient®). Other therapeuticsthat may be used are a targeted agent (e.g., a VEGF inhibitor such as amonoclonal antibody to VEGF, e.g., bevacizumab, a VEGF tyrosine kinaseinhibitor such as sorafenib, axitinib and pazopanib.

Exemplary therapeutic agents that may be used in combination with theantibodies of the invention for treatment of a chronic myelogenousleukemia (AML) include a chemotherapeutic (e.g., cytarabine,hydroxyurea, clofarabine, melphalan, thiotepa, fludarabine, busulfan,etoposide, cordycepin, pentostatin, capecitabine, azacitidine,cyclophosphamide, cladribine, topotecan), tyrosine kinase inhibitor(e.g., BCR/ABL inhibitor (e.g., imatinib, nilotinib), dual inhibitor(e.g., dasatinib, bosutinib), multikinase inhibitor (e.g., DCC-2036,ponatinib, sorafenib, sunitinib, RGB-286638), interferon alfa, steroids,apoptotic agent (e.g., omacetaxine mepesuccinat), immunotherapy (e.g.,allogeneic CD4+ memory Th1-like T cells/microparticle-boundanti-CD3/anti-CD28, autologous cytokine induced killer cells (CIK),AHN-12), CD52 targeting agent (e.g., alemtuzumab), HSP90 inhibitor(e.g., tanespimycin, STA-9090, AUY922, XL888), mTOR inhibitor (e.g.,everolimus), SMO antagonist (e.g., BMS 833923), ribonucleotide reductaseinhibitor (e.g., 3-AP), JAK-2 inhibitor (e.g., INCB018424),hydroxychloroquine, retinoid (e.g., fenretinide), cyclin-dependentkinase inhibitor (e.g., UCN-01), HDAC inhibitor (e.g., belinostat,vorinostat, JNJ-26481585), PARP inhibitor (e.g., veliparib), MDM2antagonist (e.g., RO5045337), Aurora B kinase inhibitor (e.g., TAK-901),radioimmunotherapy (e.g., actinium-225-labeled anti-CD33 antibody HuM195), Hedgehog inhibitor (e.g., PF-04449913), STAT3 inhibitor (e.g.,OPB-31121), KB004, cancer vaccine (e.g., AG858), bone marrowtransplantation, stem cell transplantation, radiation therapy, andcombinations thereof.

Exemplary therapeutic agents that may be used in combination with theantibodies of the invention for treatment of a chronic lymphocyticleukemia (CLL) include a chemotherapeutic agent (e.g., fludarabine,cyclophosphamide, doxorubicin, vincristine, chlorambucil, bendamustine,chlorambucil, busulfan, gemcitabine, melphalan, pentostatin,mitoxantrone, 5-azacytidine, pemetrexed disodium), tyrosine kinaseinhibitor (e.g., EGFR inhibitor (e.g., erlotinib), BTK inhibitor (e.g.,PCI-32765 (ibrutinib), multikinase inhibitor (e.g., MGCD265,RGB-286638), CD-20 targeting agent (e.g., rituximab, ofatumumab,RO5072759, LFB-R603), CD52 targeting agent (e.g., alemtuzumab),prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitor (e.g.,ABT-263), immunotherapy (e.g., allogeneic CD4⁺ memory Th1-like Tcells/microparticle-bound anti-CD3/anti-CD28, autologous cytokineinduced killer cells (CIK), HDAC inhibitor (e.g., vorinostat, valproicacid, LBH589, JNJ-26481585, AR-42), XIAP inhibitor (e.g., AEG35156),CD-74 targeting agent (e.g., milatuzumab), mTOR inhibitor (e.g.,everolimus), AT-101, immunotoxin (e.g., CAT-8015, anti-Tac(Fv)-PE38(LMB-2)), CD37 targeting agent (e.g., TRU-016), radioimmunotherapy(e.g., 131-tositumomab), hydroxychloroquine, perifosine, SRC inhibitor(e.g., dasatinib), thalidomide, PI3K delta inhibitor (e.g., CAL-101),retinoid (e.g., fenretinide), MDM2 antagonist (e.g., RO5045337),plerixafor, Aurora kinase inhibitor (e.g., MLN8237, TAK-901), proteasomeinhibitor (e.g., bortezomib), CD-19 targeting agent (e.g., MEDI-551,MOR208), MEK inhibitor (e.g., ABT-348), JAK-2 inhibitor (e.g.,INCB018424), hypoxia-activated prodrug (e.g., TH-302), paclitaxel or apaclitaxel agent, HSP90 inhibitor, AKT inhibitor (e.g., MK2206), HMG-CoAinhibitor (e.g., simvastatin), GNKG186, radiation therapy, bone marrowtransplantation, stem cell transplantation, and a combination thereof.

Exemplary therapeutic agents that may be used in combination with theantibodies of the invention for treatment of an acute lymphocyticleukemia (ALL) include a chemotherapeutic agent (e.g., prednisolone,dexamethasone, vincristine, asparaginase, daunorubicin,cyclophosphamide, cytarabine, etoposide, thioguanine, mercaptopurine,clofarabine, liposomal annamycin, busulfan, etoposide, capecitabine,decitabine, azacitidine, topotecan, temozolomide), tyrosine kinaseinhibitor (e.g., BCR/ABL inhibitor (e.g., imatinib, nilotinib), ON01910.Na, multikinase inhibitor (e.g., sorafenib), CD-20 targeting agent(e.g., rituximab), CD52 targeting agent (e.g., alemtuzumab), HSP90inhibitor (e.g., STA-9090), mTOR inhibitor (e.g., everolimus,rapamycin), JAK-2 inhibitor (e.g., INCB018424), HER2/neu receptorinhibitor (e.g., trastuzumab), proteasome inhibitor (e.g., bortezomib),methotrexate, asparaginase, CD-22 targeting agent (e.g., epratuzumab,inotuzumab), immunotherapy (e.g., autologous cytokine induced killercells (CIK), AHN-12), blinatumomab, cyclin-dependent kinase inhibitor(e.g., UCN-01), CD45 targeting agent (e.g., BC8), MDM2 antagonist (e.g.,RO5045337), immunotoxin (e.g., CAT-8015, DT2219ARL), HDAC inhibitor(e.g., JNJ-26481585), JVRS-100, paclitaxel or a paclitaxel agent, STAT3inhibitor (e.g., OPB-31121), PARP inhibitor (e.g., veliparib), EZN-2285,radiation therapy, steroid, bone marrow transplantation, stem celltransplantation, or a combination thereof.

Exemplary therapeutic agents that may be used in combination with theantibodies of the invention for treatment of an acute myeloid leukemia(AML) include a chemotherapeutic agent (e.g., cytarabine, daunorubicin,idarubicin, clofarabine, decitabine, vosaroxin, azacitidine,clofarabine, ribavirin, CPX-351, treosulfan, elacytarabine,azacitidine), tyrosine kinase inhibitor (e.g., BCR/ABL inhibitor (e.g.,imatinib, nilotinib), ON 01910.Na, multikinase inhibitor (e.g.,midostaurin, SU 11248, quizartinib, sorafinib), immunotoxin (e.g.,gemtuzumab ozogamicin), DT3881L3 fusion protein, HDAC inhibitor (e.g.,vorinostat, LBH589), plerixafor, mTOR inhibitor (e.g., everolimus), SRCinhibitor (e.g., dasatinib), HSP90 inhibitor (e.g., STA-9090), retinoid(e.g., bexarotene, Aurora kinase inhibitor (e.g., BI 811283), JAK-2inhibitor (e.g., INCB018424), Polo-like kinase inhibitor (e.g., BI6727), cenersen, CD45 targeting agent (e.g., BC8), cyclin-dependentkinase inhibitor (e.g., UCN-01), MDM2 antagonist (e.g., RO5045337), mTORinhibitor (e.g., everolimus), LY573636-sodium, ZRx-101, MLN4924,lenalidomide, immunotherapy (e.g., AHN-12), histamine dihydrochloride,radiation therapy, bone marrow transplantation, stem celltransplantation, and a combination thereof.

Exemplary therapeutic agents that may be used in combination with theantibodies of the invention for treatment of a multiple myeloma (MM)include a chemotherapeutic agent (e.g., melphalan, amifostine,cyclophosphamide, doxorubicin, clofarabine, bendamustine, fludarabine,adriamycin, SyB L-0501), thalidomide, lenalidomide, dexamethasone,prednisone, pomalidomide, proteasome inhibitor (e.g., bortezomib,carfilzomib, MLN9708), cancer vaccine (e.g., GVAX), CD-40 targetingagent (e.g., SGN-40, CHIR-12.12), perifosine, zoledronic acid,Immunotherapy (e.g., MAGE-A3, NY-ESO-1, HuMax-CD38), HDAC inhibitor(e.g., vorinostat, LBH589, AR-42), aplidin, cycline-dependent kinaseinhibitor (e.g., PD-0332991, dinaciclib), arsenic trioxide, CB3304,HSP90 inhibitor (e.g., KW-2478), tyrosine kinase inhibitor (e.g., EGFRinhibitor (e.g., cetuximab), multikinase inhibitor (e.g., AT9283), VEGFinhibitor (e.g., bevacizumab), plerixafor, MEK inhibitor (e.g.,AZD6244), IPH2101, atorvastatin, immunotoxin (e.g., BB-10901), NPI-0052,radioimmunotherapeutic (e.g., yttrium Y 90 ibritumomab tiuxetan), STAT3inhibitor (e.g., OPB-31121), MLN4924, Aurora kinase inhibitor (e.g.,ENMD-2076), IMGN901, ACE-041, CK-2 inhibitor (e.g., CX-4945), ananti-CD38 antibody (e.g. DARZALEX® (daratumumab), radiation therapy,bone marrow transplantation, stem cell transplantation, and acombination thereof.

Exemplary therapeutic agents that may be used in combination with theantibodies of the invention for treatment of a prostate cancer areapproved drugs for treatment of the prostate cancer, such as abirateroneacetate (Zytiga®), bicalutamide (Casodex®), cabazitaxel (Jevtana®),conjugated estrogens (Premarin®), stradiol (Estrace®), estradiolvalerate (Delestrogen®), estrogens, esterified (Menest®), degarelix(Firmagon®), docetaxel (Taxotere®), enzalutamide (Xtandi®), flutamide,goserelin acetate (Zoladex®), Cabazitaxel (Jevtana®), leuprolide acetate(Lupron®), mitoxantrone hydrochloride, nilutamide (Nilandron®)Sipuleucel-T (Provenge®) and radium 223 dichloride (Xofigo®). Otherdrugs that may be used include a chemotherapeutic agent (e.g.,carboplatin, fludarabine), hormonal therapy (e.g., cyproterone acetate,ketoconazole, aminoglutethimide, abarelix, degarelix, leuprolide,triptorelin, buserelin), tyrosine kinase inhibitor (e.g., dual kinaseinhibitor (e.g., lapatanib), multikinase inhibitor (e.g., sorafenib,sunitinib), VEGF inhibitor (e.g., bevacizumab), TAK-700, cancer vaccine(e.g., BPX-101, PEP223), lenalidomide, TOK-001, IGF-1 receptor inhibitor(e.g., cixutumumab), TRC105, Aurora A kinase inhibitor (e.g., MLN8237),proteasome inhibitor (e.g., bortezomib), OGX-011, radioimmunotherapy(e.g., HuJ591-GS), HDAC inhibitor (e.g., valproic acid, SB939, LBH589),hydroxychloroquine, mTOR inhibitor (e.g., everolimus), dovitiniblactate, diindolylmethane, efavirenz, OGX-427, genistein, IMC-3G3,bafetinib, CP-675,206, radiation therapy, surgery, or a combinationthereof.

Exemplary therapeutic agents that may be used in combination with theantibodies of the invention for treatment of a head and neck squamouscell carcinoma (HNSCC) include methotrexate (Folex®, Mexate®), bleomycin(Blenoxane®), docetaxel (Taxotere®), erbitux (Cetuximab®), hydroxyurea(Hydrea®) or pembrolizumab (Keytruda®),

In some embodiments, the antibodies of the invention are administered incombination with a TLR agonist.

In some embodiments, the TLR3 agonist is TLR4 agonist.

In some embodiments, the TLR3 agonist is a TLR7/8 agonist.

Exemplary TLR agonists are Pam3Cys, a TLR-1/2 agonist; CFA, a TLR-2agonist; MALP2, a TLR-2 agonist; Pam2Cys, a TLR-2 agonist; FSL-1, aTLR-2 agonist; Hib-OMPC, a TLR-2 agonist; polyribosinic:polyribocytidicacid (Poly I:C), a TLR-3 agonist; polyadenosine-polyuridylic acid (polyAU), a TLR-3 agonist; Polyinosinic-Polycytidylic acid stabilized withpoly-L-lysine and carboxymethylcellulose (Hiltonol®), a TLR-3 agonist;monophosphoryl lipid A (MPL), a TLR-4 agonist; LPS, a TLR-4 agonist;bacterial flagellin, a TLR-5 agonist; sialyl-Tn (STn), a carbohydrateassociated with the MUCI mucin on a number of human cancer cells and aTLR-4 agonist; imiquimod, a TLR-7 agonist; resiquimod, a TLR-7/8agonist; loxoribine, a TLR-7/8 agonist; and unmethylated CpGdinucleotide (CpG-ODN), a TLR-9 agonist.

Exemplary TLR4 agonists are agonistic antibodies specifically bindingTLR4.

In some embodiments described herein, the antibodies of the inventionare administered in combination with an antibody that bids CSF-1R

Exemplary antibodies that bind CSF-1R are those described in Int. PatentPubl. No. WO2013132044.

In some embodiments described herein, the antibodies of the inventionare administered in combination with LXRβ agonist.

In some embodiments described herein, the antibodies of the inventionare administered in combination with a DR4 agonist.

In some embodiments described herein, the antibodies of the inventionare administered in combination with a DR5 agonist.

Suitable DR4 and DR5 agonists are described for example in Int. PatentPubl. No. WO2014159562.

In some embodiments described herein, the antibodies of the inventionare administered in combination with an anti-galectin 1 antibody.

Exemplary anti-galectin 1 antibodies that may be used in combinationwith the antibodies of the invention are those described in Int. PatentPubl. No. WO2015013389.

In some embodiment described herein, the antibodies of the invention areadministered in combination with a BTK inhibitor.

In some embodiments, the BTK inhibitor is IMBRUVICA® (ibrutinib).

In some embodiments described herein, the antibodies of the inventionare administered in combination with an anti-HER2 antibody.

In some embodiments described herein, the antibodies of the inventionare administered in combination with an anti-CD20 antibody.

In some embodiments, the antibodies of the invention are administered inconjunction with (e.g., before, simultaneously or following) bone marrowtransplantation, T cell ablative therapy using chemotherapy agents suchas, fludarabine, external-beam radiation therapy (XRT),cyclophosphamide, and/or antibodies such as OKT3 or CAMPATH. In someembodiments, the antibodies of the invention may be administeredfollowing B-cell ablative therapy such as agents that react with CD20,e.g., Rituxan. For example, in one embodiment, subjects may undergostandard treatment with high dose chemotherapy followed by peripheralblood stem cell transplantation. In certain embodiments, following thetransplant, subjects receive the antibodies of the invention.

In some embodiments described herein, the antibodies of the inventionare administered before or following surgery.

In some embodiments described herein, the antibodies of the inventionare administered in combination with radiation therapy.

Radiation therapy may be administered using various methods, includingexternal-beam therapy, internal radiation therapy, implant radiation,stereotactic radiosurgery, systemic radiation therapy, radiotherapy andpermanent or temporary interstitial brachytherapy. External-beam therapyinvolves three dimensional, conformal radiation therapy where the fieldof radiation is designed, local radiation (e.g., radiation directed to apreselected target or organ), or focused radiation. Focused radiationmay be selected from stereotactic radiosurgery, fractionatedstereotactic radiosurgery or intensity-modulated radiation therapy.Focused radiation may have particle beam (proton), cobalt-60 (photon)linear accelerator (x-ray) as a radiation source (see e.g. WO2012/177624). “Brachytherapy,” refers to radiation therapy delivered bya spatially confined radioactive material inserted into the body at ornear a tumor or other proliferative tissue disease site, and includesexposure to radioactive isotopes (e.g., At-211, I-131, I-125, Y-90,Re-186, Re-188, Sm-153, Bi-212, P-32, and radioactive isotopes of Lu).Suitable radiation sources for use as a cell conditioner include bothsolids and liquids. The radiation source can be a radionuclide, such asI-125, I-131, Yb-169, Ir-192 as a solid source, I-125 as a solid source,or other radionuclides that emit photons, beta particles, gammaradiation, or other therapeutic rays. The radioactive material may alsobe a fluid made from any solution of radionuclide(s), e.g., a solutionof I-125 or I-131, or a radioactive fluid can be produced using a slurryof a suitable fluid containing small particles of solid radionuclides,such as Au-198, Y-90. The radionuclide(s) may be embodied in a gel orradioactive micro spheres.

In some embodiments, the antibodies of the invention are administered incombination with decarbazine for the treatment of melanoma. Withoutbeing bound by any particular theory, the combined use of PD-1 and/orTIM-3 blockade and chemotherapy is believed to be facilitated by celldeath that is a consequence of the cytotoxic action of mostchemotherapeutic compounds, which can result in increased levels oftumor antigen in the antigen presentation pathway. Other combinationtherapies that may result in synergy with PD-1 and/or TIM-3 blockadethrough cell death are radiation, surgery, and hormone deprivation. Eachof these protocols creates a source of tumor antigen in the host.Angiogenesis inhibitors may also be combined with PD-1 and/or TIM-3blockade Inhibition of angiogenesis leads to tumor cell death which mayfeed tumor antigen into host antigen presentation pathways.

The monospecific PD-1 and/or TIM-3 antibodies of the invention may alsobe used in combination with bispecific antibodies. Bispecific antibodiesmay be used to target two separate antigens. For example anti-Fcreceptor/anti-tumor antigen (e.g., Her-2/neu) bispecific antibodies havebeen used to target macrophages to sites of tumor. Bispecific targetingmay more effectively activate tumor specific responses. The T cell armof these responses would be augmented by the use of PD-1 and/or TIM-3blockade. Alternatively, antigen may be delivered directly to DCs by theuse of bispecific antibodies which bind to tumor antigen and a dendriticcell specific cell surface marker.

The antibodies of the invention may be used in unconjugated forms orconjugated to a second agent, e.g., a cytotoxic drug, radioisotope, or aprotein, e.g., a protein toxin or a viral protein. The antibodymolecules may be used to deliver a variety of therapeutic agents, e.g.,a cytotoxic moiety, e.g., a therapeutic drug, a radioisotope, moleculesof plant, fungal, or bacterial origin, or biological proteins (e.g.,protein toxins) or particles (e.g., a recombinant viral particles, e.g.;via a viral coat protein), or mixtures thereof.

Infectious Diseases

The invention also provides a method of treating a subject that has beenexposed to particular toxins or pathogen with the antibodies of theinvention for a time sufficient to treat the subject.

The invention also provides a method of treating a subject having aninfectious disease, comprising administering a therapeutically efficientamount of the antibody of the invention to the subject in need thereoffor a time sufficient to treat the infectious disease.

The invention also provides a method of treating a subject having aviral infection, comprising administering a therapeutically efficientamount of the antibody of the invention to the subject in need thereoffor a time sufficient to treat the viral infection.

The invention also provides a method of treating a subject having abacterial infection, comprising administering a therapeuticallyefficient amount of the antibody of the invention to the subject in needthereof for a time sufficient to treat the bacterial infection.

The invention also provides a method of treating a subject having afungal infection, comprising administering a therapeutically efficientamount of the antibody of the invention to the subject in need thereoffor a time sufficient to treat the fungal infection.

In the treatment of infection (e.g., acute and/or chronic),administration of the antibodies of the invention may be combined withconventional treatments in addition to or in lieu of stimulating naturalhost immune defenses to infection. Natural host immune defenses toinfection include inflammation, fever, antibody-mediated host defense,T-lymphocyte-mediated host defenses, including lymphokine secretion andcytotoxic T-cells (especially during viral infection), complementmediated lysis and opsonization (facilitated phagocytosis), andphagocytosis. The ability of the antibodies of the invention toreactivate dysfunctional T-cells would be useful to treat chronicinfections, in particular those in which cell-mediated immunity isimportant for complete recovery.

Similar to its application to tumors as discussed above, antibodies ofthe invention may be used alone, or as an adjuvant, in combination withvaccines, to stimulate the immune response to pathogens, toxins, andself-antigens. Examples of pathogens for which this therapeutic approachmay be useful include pathogens for which there is currently noeffective vaccine, or pathogens for which conventional vaccines are lessthan completely effective. These include HIV, Hepatitis (A, B, &®),Influenza, Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureusand Pseudomonas Aeruginosa. PD-1 and/or TIM-3 blockade may be usefulagainst established infections by agents such as HIV that presentaltered antigens over the course of the infections. These novel epitopesare recognized as foreign at the time of administration of theantibodies of the invention, thus provoking a strong T cell responsethat is not dampened by negative signals through PD-1 or TIM-3.

Viruses

For infections resulting from viral causes, the antibodies of theinvention may be combined with standard therapies for treating viralinfections. Such standard therapies vary depending upon type of virus,although in almost all cases, administration of human serum containingantibodies (e.g., IgA, IgG) specific to the virus can be effective.

Exemplary pathogenic viruses causing infections that may be treatable bythe antibodies of the invention include HIV, hepatitis (A, B, or C),herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barrvirus), adenovirus, influenza virus, flaviviruses, echovirus,rhinovirus, coxsackie virus, cornovirus, respiratory syncytial virus,mumps virus, rotavirus, measles virus, rubella virus, parvovirus,vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscumvirus, poliovirus, rabies virus, JC virus and arboviral encephalitisvirus.

In some embodiments, the virus infection is an influenza virusinfection. Influenza infection can result in fever, cough, myalgia,headache and malaise, which often occur in seasonal epidemics. Influenzais also associated with a number of postinfectious disorders, such asencephalitis, myopericarditis, Goodpasture's syndrome, and Reye'ssyndrome. Influenza infection also suppresses normal pulmonaryantibacterial defenses, such that patients recovering from influenzahave an increased risk of developing bacterial pneumonia. Influenzaviral surface proteins show marked antigenic variation, resulting frommutation and recombination. Thus, cytolytic T lymphocytes are the host'sprimary vehicle for the elimination of virus after infection. Influenzais classified into three primary types: A, B and C. Influenza A isunique in that it infects both humans and many other animals (e.g.,pigs, horses, birds and seals) and is the principal cause of pandemicinfluenza. A cell can be infected by two different influenza A strains,the segmented RNA genomes of two parental virus types mix duringreplication to create a hybrid replicant, resulting in new epidemicstrains. Influenza B does not replicate in animals and thus has lessgenetic variation and influenza C has only a single serotype.

Most conventional therapies are palliatives of the symptoms resultingfrom infection, while the host's immune response actually clears thedisease. However, certain strains (e.g., influenza A) can cause moreserious illness and death. Influenza A may be treated both clinicallyand prophylactically by the administration of the cyclic aminesinhibitors amantadine and rimantadine, which inhibit viral replication.However, the clinical utility of these drugs is limited due to therelatively high incidence of adverse reactions, their narrow anti-viralspectrum (influenza A only), and the propensity of the virus to becomeresistant. The administration of serum IgG antibody to the majorinfluenza surface proteins, hemagglutinin and neuraminidase can preventpulmonary infection, whereas mucosal IgA is required to preventinfection of the upper respiratory tract and trachea. The most effectivecurrent treatment for influenza is vaccination with the administrationof virus inactivated with formalin or β-propiolactone.

In some embodiments, the infection is a hepatitis infection, e.g., aHepatitis B or C infection.

Hepatitis B virus (HB-V) is the most infectious known blood bornepathogen. It is a major cause of acute and chronic hepatitis and hepaticcarcinoma, as well as life-long, chronic infection. Following infection,the virus replicates in hepatocytes, which also then shed the surfaceantigen HBsAg. The detection of excessive levels of HBsAg in serum isused as a standard method for diagnosing a hepatitis B infection. Anacute infection may resolve or it can develop into a chronic persistentinfection. Current treatments for chronic HBV include α-interferon,which increases the expression of class I human leukocyte antigen (HLA)on the surface of hepatocytes, thereby facilitating their recognition bycytotoxic T lymphocytes. Additionally, the nucleoside analogsganciclovir, famciclovir and lamivudine have also shown some efficacy inthe treatment of HBV infection in clinical trials. Additional treatmentsfor HBV include pegylated α-interferon, adenfovir, entecavir andtelbivudine. While passive immunity can be conferred through parentaladministration of anti-HBsAg serum antibodies, vaccination withinactivated or recombinant HBsAg also confers resistance to infection.The antibodies of the invention may be combined with conventionaltreatments for hepatitis B infections for therapeutic advantage.

Hepatitis C virus (HC-V) infection may lead to a chronic form ofhepatitis, resulting in cirrosis. While symptoms are similar toinfections resulting from Hepatitis B, in distinct contrast to HB-V,infected hosts can be asymptomatic for 10-20 years. The antibodies ofthe invention can be administered as a monotherapy, or combined with thestandard of care for hepatitis C infection. For example, the antibodiesof the invention can be administered with one or more of Sovaldi(sofosbuvir) Olysio (simeprevir), plus ribavirin or pegylatedinterferon. Although regimens that include Incivek (telaprevir) orVictrelis (boceprevir) plus ribavirin and pegylated interferon are alsoapproved, they are associated with increased side effects and longerduration of treatment.

Conventional treatment for HC-V infection includes the administration ofa combination of α-interferon and ribavirin. A promising potentialtherapy for HC-V infection is the protease inhibitor telaprevir(VX-960). Additional treatments include bavituximab (an antibody thatbinds anionic phospholipid phosphatidylserine in a B2-glycoprotein Idependent manner, Peregrine Pharmaceuticals), anti-HPV viral coatprotein E2 antibod(y)(ies) (e.g., ATL 6865-Ab68+Ab65, XTLPharmaceuticals) and Civacir® (polyclonal anti-HCV human immuneglobulin). The antibodies of the invention may be combined with one ormore of these treatments for hepatitis C infections for therapeuticadvantage. Protease, polymerase and NS5A inhibitors which may be used incombination with the antibodies of the invention to specifically treatHepatitis C infection include those described in US 2013/0045202.

In another embodiment, the infection is a measles virus. After anincubation of 9-11 days, hosts infected with the measles virus developfever, cough, coryza and conjunctivitis. Within 1-2 days, anerythematous, maculopapular rash develop, which quickly spreads over theentire body. Because infection also suppresses cellular immunity, thehost is at greater risk for developing bacterial superinfections,including otitis media, pneumonia and postinfectious encephalomyelitis.Acute infection is associated with significant morbidity and mortality,especially in malnourished adolescents.

Treatment for measles includes the passive administration of pooledhuman IgG, which can prevent infection in non-immune subjects, even ifgiven up to one week after exposure. However, prior immunization withlive, attenuated virus is the most effective treatment and preventsdisease in more than 95% of those immunized. As there is one serotype ofthis virus, a single immunization or infection typically results inprotection for life from subsequent infection.

In a small proportion of infected hosts, measles can develop into SSPE,which is a chronic progressive neurologic disorder resulting from apersistent infection of the central nervous system. S SPE is caused byclonal variants of measles virus with defects that interfere with virionassembly and budding. For these patients, reactivation of T-cells withthe antibodies of the invention so as to facilitate viral clearancewould be desirable.

In another embodiment, the infection is HIV. HIV attacks CD4⁺ cells,including T-lymphocytes, monocyte-macrophages, follicular dendriticcells and Langerhan's cells, and CD4⁺ helper/inducer cells are depleted.As a result, the host acquires a severe defect in cell-mediatedimmunity. Infection with HIV results in AIDS in at least 50% ofindividuals, and is transmitted via sexual contact, administration ofinfected blood or blood products, artificial insemination with infectedsemen, exposure to blood-containing needles or syringes and transmissionfrom an infected mother to infant during childbirth.

A host infected with HIV may be asymptomatic, or may develop an acuteillness that resembling mononucleosis—fever, headache, sore throat,malaise and rash. Symptoms can progress to progressive immunedysfunction, including persistent fever, night sweats, weight loss,unexplained diarrhea, eczema, psoriasis, seborrheic dermatitis, herpeszoster, oral candidiasis and oral hairy leukoplakia. Opportunisticinfections by a host of parasites are common in patients whoseinfections develop into AIDS.

Treatments for HIV include antiviral therapies including nucleosideanalogs, zidovudine (AST) either alone or in combination with didanosineor zalcitabine, dideoxyinosine, dideoxycytidine, lamidvudine, stavudine;reverse transcriptive inhibitors such as delavirdine, nevirapine,loviride, and proteinase inhibitors such as saquinavir, ritonavir,indinavir and nelfinavir. Treatments for HIV include EDURANT®(rilpivirine). The antibodies of the invention may be combined withconventional treatments for HIV infections for therapeutic advantage.

In another embodiment, the infection is a Cytomegalovirus (CMV)infection. CMV infection is often associated with persistent, latent andrecurrent infection. CMV infects and remains latent in monocytes andgranulocyte-monocyte progenitor cells. The clinical symptoms of CMVinclude mononucleosis-like symptoms (i.e., fever, swollen glands,malaise), and a tendency to develop allergic skin rashes to antibiotics.The virus is spread by direct contact. The virus is shed in the urine,saliva, semen and to a lesser extent in other body fluids. Transmissioncan also occur from an infected mother to her fetus or newborn and byblood transfusion and organ transplants. CMV infection results ingeneral impairment of cellular immunity, characterized by impairedblastogenic responses to nonspecific mitogens and specific CMV antigensand diminished cytotoxic ability.

Treatments of CMV infection include the anti-virals ganciclovir,foscarnet and cidovir, but these drugs are typically only prescribed inimmunocompromised patients. The antibodies of the invention describedherein may be combined with conventional treatments for cytomegalovirusinfections for therapeutic advantage.

In another embodiment, the infection is Epstein-Barr virus (EBV)infection. EBV can establish persistent and latent infections andprimarily attacks B cells. Infection with EBV results in the clinicalcondition of infectious mononucleosis, which includes fever, sorethroat, often with exudate, generalized lymphadenopathy andsplenomegaly. Hepatitis is also present, which can develop intojaundice.

While typical treatments for EBV infections are palliative of symptoms,EBV is associated with the development of certain cancers such asBurkitt's lymphoma and nasopharyngeal cancer. Thus, clearance of viralinfection before the complications develop would be of great benefit.The antibodies of the invention may be combined with conventionaltreatments for Epstein-Barr virus infections for therapeutic advantage.

In another embodiment, the infection is Herpes simplex virus (HSV)infection. HSV is transmitted by direct contact with an infected host. Adirect infection may be asymptomatic, but typically result in blisterscontaining infectious particles. The disease manifests as cycles ofactive periods of disease, in which lesions appear and disappear as thevirus latently infects the nerve ganglion for subsequent outbreaks.Lesions may be on the face, genitals, eyes and/or hands. In some case,an infection can also cause encephalitis.

Treatments for herpes infections are directed primarily to resolving thesymptomatic outbreaks, and include systemic antiviral medicines such as:acyclovir (e.g., Zovirax®), valaciclovir, famciclovir, penciclovir, andtopical medications such as docosanol (Abreva®), tromantadine andzilactin. The clearance of latent infections of herpes would be of greatclinical benefit. The antibodies of the invention may be combined withconventional treatments for herpes virus infections for therapeuticadvantage.

In another embodiment, the infection is Human T-lymphotrophic virus(HTLV-1, HTLV-2). HTLV is transmitted via sexual contact, breast feedingor exposure to contaminated blood. The virus activates Th1 cells,resulting in their overproliferation and overproduction of Th1 relatedcytokines (e.g., IFN-γ and TNF-α). This in turn results in a suppressionof Th2 lymphocytes and reduction of Th2 cytokine production (e.g., IL-4,IL-5, IL-10 and IL-13), causing a reduction in the ability of aninfected host to mount an adequate immune response to invading organismsrequiring a Th2-dependent response for clearance (e.g., parasiticinfections, production of mucosal and humoral antibodies).

HTLV infections lead to opportunistic infections resulting inbronchiectasis, dermatitis and superinfections with Staphylococcus spp.and Strongyloides spp. resulting in death from polymicrobial sepsis.HTLV infection can also lead directly to adult T-cell leukemia/lymphomaand progressive demyelinating upper motor neuron disease known asHAM/TSP. The clearance of HTLV latent infections would be of greatclinical benefit. The antibodies of the invention may be combined withconventional treatments for HTLV infections for therapeutic advantage.

In another embodiment, the infection is Human papilloma virus (HPV). HPVprimarily affects keratinocytes and occurs in two forms: cutaneous andgenital. Transmission is believed to occur through direct contact and/orsexual activity. Both cutaneous and genital HPV infection can result inwarts and latent infections and sometimes recurring infections, whichare controlled by host immunity which controls the symptoms and blocksthe appearance of warts, but leaves the host capable of transmitting theinfection to others.

Infection with HPV can also lead to certain cancers, such as cervical,anal, vulvar, penile and oropharynial cancer. There are no known curesfor HPV infection, but current treatment is topical application ofImiquimod, which stimulates the immune system to attack the affectedarea. The clearance of HPV latent infections would be of great clinicalbenefit. The antibodies of the invention may be combined withconventional treatments for HPV infections for therapeutic advantage.

Bacterial Infections

Some examples of pathogenic bacteria causing infections that may betreated with the antibodies of the invention include syphilis,chlamydia, rickettsial bacteria, mycobacteria, staphylococci,streptococci, pneumonococci, meningococci and conococci, klebsiella,proteus, serratia, pseudomonas, legionella, diphtheria, salmonella,bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, andLymes disease bacteria. The antibodies of the invention can be used incombination with existing treatment modalities for the aforesaidinfections. For example, treatments for syphilis include penicillin (eg, penicillin G), tetracycline, doxycycline, ceftriaxone andazithromycin.

Lyme disease, caused by Borrelia burgdorferi is transmitted into humansthrough tick bites. The disease manifests initially as a localized rash,followed by flu-like symptoms including malaise, fever, headache, stiffneck and arthralgias. Later manifestations can include migratory andpolyarticular arthritis, neurologic and cardiac involvement with cranialnerve palsies and radiculopathy, myocarditis and arrhythmias. Some casesof Lyme disease become persistent, resulting in irreversible damageanalogous to tertiary syphilis. Current therapy for Lyme diseaseincludes primarily the administration of antibiotics.Antibiotic-resistant strains may be treated with hydroxychloroquine ormethotrexate. Antibiotic refractory patients with neuropathic pain canbe treated with gabapentin. Minocycline may be helpful in late/chronicLyme disease with neurological or other inflammatory manifestations.

Other forms of borreliois, such as those resulting from B. recurentis,B. hermsii, B. turicatae, B. parikeri, B. hispanica, B. duttonii and B.persica, as well leptospirosis (E.g., L. interrogans), typically resolvespontaneously unless blood titers reach concentrations to causeintrahepatic obstruction.

Fungi and Parasites

Some examples of pathogenic fungi causing infections that may be treatedwith the antibodies of the invention include Candida (albicans, krusei,glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus(fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus),Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioidesbrasiliensis, Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable withthe antibodies of the invention described herein include Entamoebahistolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp.,Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodiumvivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi,Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.

Diagnostic Uses and Kits

Kits

The invention also provides a kit comprising the antagonistic antibodyspecifically binding PD-1 of the invention.

The invention also provides a kit comprising the antagonistic antibodyspecifically binding TIM-3 of the invention.

The invention also provides a kit comprising the antagonistic bispecificPD-1/TIM-3 antibody comprising a first domain specifically binding PD-1and a second domain specifically binding TIM-3 of the invention.

The kit may be used for therapeutic uses and as diagnostic kits.

The kit may be used to detect the presence of PD-1, TIM-3, or PD-1 andTIM-3 in a biological sample.

In some embodiments, the kit comprises the antibody of the inventiondescribed herein and reagents for detecting the antibody. The kit caninclude one or more other elements including: instructions for use;other reagents, e.g., a label, a therapeutic agent, or an agent usefulfor chelating, or otherwise coupling, an antibody to a label ortherapeutic agent, or a radioprotective composition; devices or othermaterials for preparing the antibody for administration;pharmaceutically acceptable carriers; and devices or other materials foradministration to a subject.

In some embodiments, the kit comprises the antibody of the invention ina container and instructions for use of the kit.

In some embodiments, the antibody in the kit is labeled.

In some embodiments, the kit comprises the antagonistic antibodyspecifically binding PD-1, comprising

the VH of SEQ ID NO: 41 and the VL of SEQ ID NO: 49;

the VH of SEQ ID NO: 41 and the VL of SEQ ID NO: 50;

the VH of SEQ ID NO: 42 and the VL of SEQ ID NO: 51;

the VH of SEQ ID NO: 42 and the VL of SEQ ID NO: 52;

the VH of SEQ ID NO: 42 and the VL of SEQ ID NO: 53;

the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 49;

the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 54;

the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 50;

the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 55;

the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 56;

the VH of SEQ ID NO: 43 and the VL of SEQ ID NO: 57;

the VH of SEQ ID NO: 44 and the VL of SEQ ID NO: 49;

the VH of SEQ ID NO: 45 and the VL of SEQ ID NO: 49;

the VH of SEQ ID NO: 46 and the VL of SEQ ID NO: 49;

the VH of SEQ ID NO: 47 and the VL of SEQ ID NO: 49;

the VH of SEQ ID NO: 48 and the VL of SEQ ID NO: 53;

the VH of SEQ ID NO: 48 and the VL of SEQ ID NO: 52;

the VH of SEQ ID NO: 48 and the VL of SEQ ID NO: 56;

the VH of SEQ ID NO: 47 and the VL of SEQ ID NO: 58;

the VH of SEQ ID NO: 47 and the VL of SEQ ID NO: 59;

the VH of SEQ ID NO: 45 and the VL of SEQ ID NO: 60;

the VH of SEQ ID NO: 45 and the VL of SEQ ID NO: 61;

the VH of SEQ ID NO: 45 and the VL of SEQ ID NO: 62;

the VH of SEQ ID NO: 63 and the VL of SEQ ID NO: 65; or

the VH of SEQ ID NO: 64 and the VL of SEQ ID NO: 65.

In some embodiments, the kit comprises the antagonistic antibodyspecifically binding PD-1 comprising the VH of SEQ ID NO: 48 and the VLof SEQ ID NO: 56.

In some embodiments, the kit comprises the antagonistic antibodyspecifically binding PD-1 comprising the VH of SEQ ID NO: 64 and the VLof SEQ ID NO: 65.

In some embodiments, the kit comprises the antagonistic antibodyspecifically binding TIM-3, comprising

the VH of SEQ ID NO: 145 and the VL of SEQ ID NO: 155;

the VH of SEQ ID NO: 146 and the VL of SEQ ID NO: 156;

the VH of SEQ ID NO: 148 and the VL of SEQ ID NO: 157;

the VH of SEQ ID NO: 147 and the VL of SEQ ID NO: 155;

the VH of SEQ ID NO: 149 and the VL of SEQ ID NO: 158;

the VH of SEQ ID NO: 150 and the VL of SEQ ID NO: 159;

the VH of SEQ ID NO: 151 and the VL of SEQ ID NO: 160;

the VH of SEQ ID NO: 152 and the VL of SEQ ID NO: 161;

the VH of SEQ ID NO: 153 and the VL of SEQ ID NO: 162;

the VH of SEQ ID NO: 154 and the VL of SEQ ID NO: 163; or

the VH of SEQ ID NO: 172 and the VL of SEQ ID NO: 173.

In some embodiments, the kit comprises the antagonistic antibodyspecifically binding TIM-3 comprising the VH of SEQ ID NO: 146 and theVL of SEQ ID NO: 156.

In some embodiments, the kit comprises the antagonistic antibodyspecifically binding TIM-3 comprising the VH of SEQ ID NO: 172 and theVL of SEQ ID NO: 173.

In some embodiments, the kit comprises the antagonistic bispecificPD-1/TIM-3 antibody comprising the HC1, the LC1, the HC2 and the LC2 of

SEQ ID NOs: 186, 188, 190 and 193, respectively;

SEQ ID NOs: 186, 188, 191 and 194, respectively;

SEQ ID NOs: 187, 189, 190 and 193, respectively;

SEQ ID NOs: 187, 189, 191, 194, respectively;

SEQ ID NOs: 186, 188, 192 and 195, respectively;

SEQ ID NOs: 186, 188, 248 and 194, respectively;

SEQ ID NOs: 241, 188, 244, 195, respectively;

SEQ ID NOs: 241, 188, 245, 194, respectively;

SEQ ID NOs: 242, 189, 246, 194, respectively;

SEQ ID NOs: 243, 188, 246, 194, respectively; or

SEQ ID NOs: 243, 188, 247, 195, respectively.

Methods of Detecting PD-1, TIM-3 or PD-1 and TIM-3

The invention also provides a method of detecting PD-1 in a sample,comprising obtaining the sample, contacting the sample with theantagonistic antibody specifically binding PD-1 of the invention, anddetecting the antibody bound to PD-1 in the sample.

The invention also provides a method of detecting TIM-3 in a sample,comprising obtaining the sample, contacting the sample with theantagonistic antibody specifically binding TIM-3 of the invention, anddetecting the antibody bound to TIM-3 in the sample.

The invention also provides a method of detecting PD-1 and TIM-3 in asample, comprising obtaining the sample, contacting the sample with theantagonistic bispecific PD-1/TIM-3 antibody comprising a first domainspecifically binding PD-1 and a second domain specifically binding TIM-3of the invention, and detecting the antibody bound to PD-1 and TIM-3 inthe sample.

In some embodiments, the sample may be derived from urine, blood, serum,plasma, saliva, ascites, circulating cells, circulating tumor cells,cells that are not tissue associated (i.e., free cells), tissues (e.g.,surgically resected tumor tissue, biopsies, including fine needleaspiration), histological preparations, and the like.

The antibodies of the invention bound to PD-1, TIM-3 or PD-1 and TIM-3may be detected using known methods. Exemplary methods include directlabeling of the antibodies using fluorescent or chemiluminescent labels,or radiolabels, or attaching to the antibodies of the invention a moietywhich is readily detectable, such as biotin, enzymes or epitope tags.Exemplary labels and moieties are ruthenium, ¹¹¹In-DOTA,¹¹¹In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase,alkaline phosphatase and beta-galactosidase, poly-histidine (HIS tag),acridine dyes, cyanine dyes, fluorone dyes, oxazin dyes, phenanthridinedyes, rhodamine dyes and Alexafluor® dyes.

The antibodies of the invention may be used in a variety of assays todetect PD-1, TIM-3 or PD-1 and TIM-3 in the sample. Exemplary assays arewestern blot analysis, radioimmunoassay, surface plasmon resonance,immunoprecipitation, equilibrium dialysis, immunodiffusion,electrochemiluminescence (ECL) immunoassay, immunohistochemistry,fluorescence-activated cell sorting (FACS) or ELISA assay.

The present invention will now be described with reference to thefollowing specific, non-limiting examples.

Example 1. General Methods

Purified Human Mixed Lymphocyte Reaction (MLR)

A purified human mixed lymphocyte reaction (MLR assay) was used tomeasure changes in cytokine production induced by addition of testantibodies to co-cultures of CD4⁺ T cells and dendritic cells.

Peripheral blood mononuclear cells (PBMCs) were isolated from a leukopak(Biological Specialty Corporation) using a Ficoll gradient. CD4⁺ T cellswere then freshly isolated by negative selection from PBMCs using theMiltenyi AutoMACS and CD4⁺ T cell isolation beads per manufacturer'sinstructions or were commercially purchased as frozen CD4⁺ T cells(Hemacare Corporation). One dendritic cell donor (Hemacare Corporation)was used. Post-isolation or thaw, CD4⁺ T cells and dendritic cells werewashed and resuspended in assay media (RMPI1640 media supplemented with10% fetal bovine serum, 1% penicillin/streptomycin, 1× non-essentialamino acids, and 1× sodium pyruvate-Invitrogen). The purified human CD4⁺T cells were diluted to 1×10⁶ cells/mL and seeded at 100,000 cells/100μL/well. Dendritic cells were diluted to 0.1×10⁶ cells/mL and seeded at5,000 cells/50 μL/well in U-bottom plates. Test antibodies or controlantibodies were prepared at a 4× concentration in assay media yielding1× when 50 μL of antibody was added to 150 μL of cells.

10-point serial dilutions of test or control antibodies were added tothe wells at a final concentration of: 30, 10, 3.33, 1.11, 0.37, 0.12,0.04, 0.01, 0.0046 and 00015 nM. CD4⁺ T cells plus dendritic cells anddendritic cells alone were included as controls to measure basalcytokine secretion. Cells were maintained at 37° C., 5% CO₂ for 5 days.On Day 5, 100 μL of tissue culture supernatant was removed from cultureplates and transferred to V-bottom plates. Supernatant was frozen atleast overnight at −80° C. Cumulative cytokine production was measuredin tissue culture supernatant using Meso Scale Discovery (MSD) Th1/Th2human cytokine 10-plex plates following manufacturer's protocol.Briefly, MSD plates were blocked with 1% blocker B overnight at 4° C.The following day, blocker was removed and plates were washed using theBiotek 406 plate washer. An 8-point standard curve were prepared andadded in duplicate to the plates. Thawed tissue culture supernatant wasadded at 25 μL/well, plates were sealed and shaken vigorously for 1.5hours. Without removing standards or supernatant, 25 pt of detectionantibody was added to each well. Plates were sealed, and shakenvigorously for 1.5 hours. Plates were washed, read buffer was added andplates were read using Meso Scale Discovery's plate reader.

Cytokine concentrations were calculated by MSD software. Theconcentration of cytokine in unknown samples is calculated by comparingthe unknown's output signal to the output signal and known cytokineconcentrations in the standard curve. Calculated concentrations wereuploaded in Spotfire TIBCO software for visualization. After a visualinspection of the data, MAD-median outlier procedure with a threshold of3.5 was used to identify and exclude outliers on log-transformed data.Robust analysis of the half-maximal effective concentration (RobustEC50) was carried out on each cytokine for each antibody.

CMV Assay

A cytomegalovirus antigen recall assay (CMV assay) was used to measurechanges in cytokine production induced by addition of test antibodies tocultures of peripheral blood mononuclear cells (PBMCs) with CMV wholeantigen (for PD-1 antibodies) or with a pool of 138 15-mer peptides thatoverlap through the 65 kd phosphoprotein (pp65) (for TIM-3 mAbs andPD1/TIM-3 bispecific mAbs).

Post-thaw, PBMCs (Astarte Biologics and Hemcare Corporation) were washedand resuspended in assay media (RMPI1640 media supplemented with 10%fetal bovine serum, 1% penicillin/streptomycin, 1× non-essential aminoacids, and 1× sodium pyruvate-Invitrogen). The PBMCs were diluted to1.5×10⁶ cells/mL and seeded at 150,000 cells/100 CMV antigen (AstarteBiologics) was prepared at a 4× concentration of 0.4 μg/mL in assaymedia yielding 0.1 μg/mL when 50 μL of antigen was added to 100 μL ofcells and 50 μL of antibody. Antibodies were prepared at a 4×concentration in assay media yielding 1× when 50 μL of antibody wasadded to cells and peptide.

Serial dilutions of test antibodies were added to the wells at a finalconcentration between 150-0.001 nM. Cells plus CMV antigen or pp65 pool,cells alone, and isotype control prepared at a final concentration of 50or 30 nM were included as controls to measure basal cytokine secretion.Cells were maintained at 37° C., 5% CO₂ for 6 days. For MSD analysis, onDay 6, 100 μL of tissue culture supernatant was removed from cultureplates and transferred to V-bottom plates. Supernatant was frozen atleast overnight at −80° C. Cumulative cytokine production was measuredin tissue culture supernatant using Meso Scale Discovery (MSD) Th1/Th2human cytokine 10-plex plates following manufacturer's protocol.Briefly, MSD plates were blocked with 1% blocker B overnight at 4° C.The following day, blocker was removed and plates were washed using theBiotek 406 plate washer. An 8-point standard curve was prepared andadded in duplicate to the plates. Thawed tissue culture supernatant wasadded at 25 μL/well, plates were sealed and shaken vigorously for 1.5hours. Without removing standards or supernatant, 25 μL of detectionantibody was added to each well. Plates were sealed, and shakenvigorously for 1.5 hours. Plates were washed, read buffer was added andplates were read using Meso Scale Discovery's plate reader.

Cytokine concentrations were calculated by MSD software. Theconcentration of cytokine in unknown samples is calculated by comparingthe unknown's output signal to the output signal and known cytokineconcentrations in the standard curve. Calculated concentrations wereuploaded in Spotfire TIBCO software for visualization. After a visualinspection of the data, MAD-median outlier procedure with a threshold of3.5 was used to identify and exclude outliers on log-transformed data.Robust analysis of the half-maximal effective concentration (RobustEC50) was carried out on each cytokine for each antibody.

For TIM-3 antibodies and PD1/TIM-3 bispecific antibodies, at day 6,after supernatant was collected for MSD analysis, cells were washed oncewith PBS and subsequently stained for Live/Dead discrimination and thefollowing cell surface markers: CD3, CD4, CD8, CD137, PD-1 and TIM-3.Flow cytometry was performed on a LSR Fortessa (BD). Data was analyzedusing the Flow Jo software. CD137+ cells were identified based onFluorescence Minus One (FMO) method on viable CMV-treated CD8+ and CD4+cells.

For the sequential treatment experiments, CMV recall assays were carriedout as above with pp65 peptide pool stimulation for six days. On daysix, supernatant was removed and cells were restimulated with pp65 poolin the presence of anti-TIM-3 antibodies. Twenty-four hours later,supernatant was removed and IFN-γ levels were measured by MSD, asdescribed above.

PD-1 Ligand Inhibition Assay

The ligand inhibition assay design was MSD (Mescoscale Discovery) based.A MSD plate was directly coated with ligand (cynoPDL1-ECD, huPDL1-ECD orhuPDL2-ECD) and incubated overnight at 4° C. The following day, thecoating solution was removed and the plate was blocked. A fixedconcentration of biotinylated PD-1 (huPD1-ECD) was pre incubated withantibodies or with an isotype control antibody as a negative control.Depending on the panel of antibodies to be tested, the antibodies weretested as titrations or at a fixed concentration. The MSD plate waswashed and the biotinylated PD-1/antibody mixture was added to theligand coated MSD plate. The plate was washed and biotinylated PD-1bound to ligand was detected by ruthenylated streptavidin Inhibition ofPD-1 binding by an antibody resulted in decreased signal in the MSDassay. Maximal biotinylated PD-1 binding in the absence of inhibitor wasdetermined and sometimes used to normalize the data to a percentage ofmaximal biotinylated PD-1 signal. The mAbs that were positive forinhibition of ligand binding at one concentration were also tested indose responses for inhibition of various PD-1 ligands.

Jurkat Cell Binding

Jurkat cells were stimulated overnight with 20 ng/ml of PHA, harvested,washed, and checked for viability. The cells were then incubated at6-10° C. for 45-60 minutes with various concentrations of testantibodies, washed and incubated at 6-10° C. for 45-60 minutes withFITC-labeled goat anti-human IgG. The cells were washed and fixed withBD Cytofix, refrigerated overnight and analyzed on a MACSQuant flowcytometer. The percentage of PD-1 positive cells at each antibodyconcentration was plotted vs log of the antibody concentration and EC₅₀values were generated in Prism.

Affinity Measurements

PD-1 mAbs

Anti-PD-1 mAbs were tested for binding affinity to huPD1-ECD andcynoPD-1-ECD. Affinity measurements using Surface Plasmon Resonance(SPR) were performed using a ProteOn XPR36 system. A biosensor surfacewas prepared by coupling a mixture of anti-IgG Fc modified alginatepolymer layer surface of a GLC chip using the manufacturer instructionsfor amine-coupling chemistry. Test mAbs were captured and theirinteractions with analytes (huPD1-ECD or cynoPD1-ECD) were monitored inPBS-based buffer at 25° C. The collected data were processed and fittedto a Langmuir 1:1 binding model. The result for each mAb was reported inthe format of k_(on) (On-rate), k_(off) (Off-rate) and K_(D)(equilibrium dissociation constant).

TIM-3 Ligand Inhibition Assay

TIM-3/galectin-9 competition ELISAs were done by binding 1μg/mlrecombinant human Fc-TIM-3 chimera (R&D Systems-cat #: 2365-TM-05)in PBS per well of a 96-well White Maxisorp plate (Nunc). The plateswere washed and blocked with StartingBlock T20 (Pierce) and inhibitor ata 10 μg/ml concentration was added to the wells. Without washing, 7.5μg/ml galectin-9 at was added to the wells and incubated for 30 minAnti-galectin-9-biotin antibody polyclonal antibody (R&D Systems) at 0.5μg/mL was then added and incubated for 30 minutes. The plates werewashed and neutravidin-HRP-conjugated (Pierce) was added and the platesincubated for an additional 45 minutes. The plates were washed and PODChemiluminescence substrate (Roche) was added immediately prior toreading plates and the luminescence was read on a luminometer.

Generation of Antigens Used in the Study

Cloning, expression and purification of the antigens was done usingstandard methods. Various protein fragments were expressed ashexahistidine tag or Fc fusion proteins. The amino acid sequences of theused proteins without the tag sequences are shown in SEQ ID NOs: 1-9,138 and 89.

Full length human PD1 (huPD1); SEQ ID NO: 1PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADG PRSAQPLRPE DGHCSWPLExtracellular domain of human PD1 (huPD1-ECD); SEQ ID NO: 2PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLMacaca fascicularis (cynomolgous, hereinreferred to as cyno) PD1 (cPD1); SEQ ID NO: 3)PGWFLESPDRPWNAPTFSPALLLVTEGDNATFTCSFSNASESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTRLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQALVVGVVGGLLGSLVLLVWVLAVICSRAAQGTIEARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPAPCVPEQTEYATIVFPSGLGTSSPARRGSADG PRSPRPLRPEDGHCSWPLExtracellular domain of cyno PD1 (cPD1-ECD); SEQ ID NO: 4PGWFLESPDRPWNAPTFSPALLLVTEGDNATFTCSFSNASESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTRLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQALFull length human PD-L1 (huPD-L1); SEQ ID NO: 5FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEE NHTAELVIPELPLAHPPNERExtracellular domain of human PD-L1 (huPDL1-ECD) SEQ ID NO: 6FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEE NHTAELVIPELPLAHPPNERTExtracellular domain of cynomolgus PD-L1 (cynoPDL1-ECD) SEQ ID NO: 7AFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLTSLIVYWEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCIFRRLDPEENHTAELVIPELPLALPPNERT Extracellular domain of human PD-L2 (huPDL2-ECD)SEQ ID NO: 8 LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHP TExtracellular domain of mouse PD1 (musPD1-ECD) SEQ ID NO: 9LEVPNGPWRSLTFYPAWLTVSEGANATFTCSLSNWSEDLMLNWNRLSPSNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIEESPGAELVVTERILETSTRYPSPSPKPEGRFQ Full length human TIM-3,SEQ ID NO: 138 SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAMP Extracellular domain of human TIM-3(huTIM-3-ECD) SEQ ID NO: 89SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIR

Example 2. Selection of Human Anti-PD-1 Antibodies from Phage DisplayLibraries

PD-1 binding Fabs were selected from de novo pIX phage display librariesas described in Shi et al., J Mol Biol 397:385-96, 2010, Int. PatentPubl. No. WO2009/085462 and U.S. Patent Publ. No. US2010/0021477.Briefly, the libraries were generated by diversifying human scaffoldswhere germline VH genes IGHV1-69*01, IGHV3-23*01, and IGHV5-51*01 wererecombined with the human IGHJ-4 minigene via the H3 loop, and humangermline VL kappa genes 012 (IGKV1-39*01), L6 (IGKV3-11*01), A27(IGKV3-20*01), and B3 (IGKV4-1*01) were recombined with the IGKJ-1minigene to assemble complete VH and VL domains. The positions in theheavy and light chain variable regions around H1, H2, L1, L2 and L3loops corresponding to positions identified to be frequently in contactwith protein and peptide antigens were chosen for diversification.Sequence diversity at selected positions was limited to residuesoccurring at each position in the IGHV or IGLV germline gene families ofthe respective IGHV or IGLV genes. Diversity at the H3 loop wasgenerated by utilizing short to mid-sized synthetic loops of lengths7-14 amino acids. The amino acid distribution at H3 was designed tomimic the observed variation of amino acids in human antibodies. Librarydesign is detailed in Shi et al., (2010) J Mol Biol 397:385-96. Thescaffolds utilized to generate libraries were named according to theirhuman VH and VL germline gene origin. The three heavy chain librarieswere combined with the four germline light chains or combined with thediversified light chain libraries to generate 12 unique VH: VLcombinations. These libraries were later combined further based onlibrary versions to generate additional libraries for panningexperiments against PD-1.

The libraries were panned against huPD1-ECD, cynoPD1-ECD, musPD1-ECD,huPD1-Fc and/or musPD1-Fc. The recombinant proteins were biotinylated(bt) and captured on streptavidin magnetic beads (Dynal), then exposedto the de novo pIX Fab libraries at a final concentration of 100 nM or10 nM. Non-specific phages were washed away in PBS-Tween and boundphages were recovered by infection of MC1061F′ E. coli cells. Phageswere amplified from these cells overnight and panning was repeated for atotal of three or four rounds. Following the final round of biopanning,monoclonal Fab was screened for binding to huPD1-ECD, huPD1-Fc,musPD1-Fc and/or cynoPD1-Fc in two ELISA formats. In Format 1, Fab wascaptured on an ELISA plate by anti-Fd antibody and the various forms ofbtPD1's were added to captured Fab, followed by detection of bt-PD1'swith Streptavidin:HRP. In Format 2, the various forms of btPD1's werecaptured on ELISA plates by Streptavidin and secreted Fab was added tothe captured antigen, followed by detection of the Fab withGoatAntiFab'2HRP. Clones that demonstrated binding to the proteins weresequenced in the heavy and light chain variable regions.

Fabs from the human PD-1 or mouse PD-1 selections were then tested forcross-reactivity to cynoPD1-Fc secreted in mammalian cell supernatant.Fab was captured on an ELISA plate by anti-Fd antibody and thecynoPD1-Fc supernatant was added to the captured Fab, followed bydetection of cynoPD1-Fc with GoatAntiHumanFc:HRP. Based on bindingcharacteristics to cynoPD1-Fc, select antibodies were chosen for furthercharacterization.

Select Fabs were chosen for further characterization and were cloned asIgG2sigma/κ. IgG2sigma has abolished effector functions and has V234A,G237A, P238S, H268A, V309L, A330S and P331S substitutions when comparedto the wild type IgG2. IgG2sigma is described in U.S. Pat. No.8,961,967. The antibodies were evaluated for their ability to blockhuman PD-1 binding to cynomolgus PD-L1, affinity to human and cynomolgusPD-1 proteins, and their ability to bind to cells endogenouslyexpressing human PD-1 (Jurkat cells). The antibodies were subsequentlyevaluated for their ability to block human PD-L1 and human PD-L2 bindingto huPD1.

Based on the results, several antibodies were chosen for affinitymaturation. Characteristics of select antibodies chosen for affinitymaturation are shown in Table 7.

TABLE 7 Ligand inhibition; IC₅₀ (μg/ml) Jurkat binding; ProteOn SPRaffinity mAb cynoPD-L1 huPD-L1 huPD-L2 EC₅₀ μg/ml k_(on) (1/Ms) k_(off)(1/s) K_(D) (nM) PD1B11 0.017-0.018 0.019 0.029 0.03-0.24 4.68E+058.96E−03 19.2 PD1B70 0.010-0.021 0.040 0.059 0.69-1.32 1.84E+05 3.04E−02166 PD1B71 0.014-0.015 0.024 0.035 0.13-0.47 2.31E+05 2.77E−02 120 Hu:human Cyno: cynomolgus

Example 3. Affinity-Maturation of Human Anti-PD-1 Antibodies

Antibodies PD1B70, PD1B71 and PD1B114 (close homolog to PD1B11), wereaffinity matured in Fab format using phage display libraries withdiversity at select VL positions and at HCDR1 and HCDR2. The design ofaffinity-maturation libraries for each Fab is shown in Table 8. Residuenumbering is according to PD1B114 VH SEQ ID NO: 41 in Table 8.

TABLE 8 Position Parent amino acid Residues used for diversificationDiversification of PD1B114, PD1B70 and PD1B71 VH 30 S D, K, S 31 S D, N,S, T 32 Y A, D, S, Y 33 A A, D, G, S, W, Y 35 S H,N, S 50 G A, E, G, N,R, T, W, Y 52 I A, D, I, N, R, S 54 I E, I, N, S, Y 55 F E, F, Q, S, Y57 T D, N, R, S, T, Y 59 N E, G, N, Q, R, Y Diversification of PD1B114,PD1B70 and PD1B71 VL 30 S D, N, R, S 31 S N, S, T 32 Y D, N, R, S, Y 49Y E, H, K, Y 50 D D, G, S, W, Y 53 N D, N, S, T, Y 91 R A, D, E, G, H,N, R, S, W, Y 92 S A, D, E, G, H, N, R, S, W, Y 93 N A, D, E, G, H, N,R, S, W, Y 94 W A, D, E, G, H, N, R, S, W, Y 96 L F, I, L, N, R, W, Y

The libraries were constructed and phage was generated. The VH and theVL phage libraries were then used for phage panning against huPD1-ECDand cynoPD1-ECD biotinylated recombinant proteins. Following phagepanning, soluble Fabs were screened for binding to both human and cynoPD-1. Select Fabs were cloned as IgG2sigma isotype and characterized fortheir Jurkat cell binding and cynomolgus PD-L1 ligand inhibition atconcentrations 1 μg/ml and 10 μg/ml.

Table 9 shows the characterization results of the parental andaffinity-matured antibodies.

TABLE 9 Ligand inhibition at indicated Jurkat Cell concentration*binding; EC₅₀ mAb 1 μg/ml 10 μg/ml (μg/ml) PD1B11 5% 5% 0.05 PD1B114 8%13%  0.47 PD1B149 7% 7% 0.08 PD1B160 4% 3% 0.08 PD1B162 7% 6% 0.05PD1B164 6% 3% 0.06 PD1B183 5% 5% 0.08 PD1B184 4% 4% 0.08 PD1B185 8% 5%0.09 PD1B187 7% 5% 0.09 PD1B192 5% 5% 0.06 PD1B70 6% 6% 0.69 PD1B175 6%5% 0.09 PD1B71 6% 9% 0.13 PD1B177 7% 8% 0.05 *value indicates percentageligand not blocked

The affinity matured antibodies were assessed in affinity experiments asdescribed above using ProteOn SPR analyses for binding to huPD1-ECD andcynoPD1-ECD. The binding characteristics of the mAbs to cyno PD-1 areshown in Table 10 and to human PD-1 in Table 11. STDEV were calculatedfor 3 or more replicates generated for human and cyno proteins. If lessthan 3 replicates were calculated, RANGE was indicated. RANGE is definedas the low and high values for the replicates tested. For samples in theTable 10 or Table 11 without value indicated in RANGE or STDEV, only oneexperiment was performed. The best affinity matured variants hadaffinities for human and cyno PD-1 in the single digit nM rangefollowing ˜4-20 fold gains in affinity compared to their parental mAbs.

TABLE 10 antigen: cyno PD-1 k_(on) STDEV. kon k_(off) STDEV. koff K_(D)STDEV. K_(D) Sample (1/Ms) or RANGE (1/s) or RANGE (nM) or RANGE PD1B702.10E+05 (1.99-2.25)E+05 2.58E−02 (2.45-2.75)E−02 123 109-138 PD1B1752.14E+05 (1.98-2.30)E+05 6.40E−03 (6.06-6.73)E−03 30 26-34 PD1B713.04E+05 2.35E+04 2.03E−02 7.27E−04 66.8 5.68 PD1B177 2.92E+05(2.80-3.04)E+05 1.89E−03 (1.84-1.93)E−03 6.47 6.1-6.9 PD1B114 2.94E+051.69E+04 2.39E−02 1.45E−03 81.5 6.8  PD1B149 3.20E+05 (3.04-3.36)E+053.57E−03 (3.48-3.65)E−03 11.2 (10.9-11.4) PD1B160 3.17E+05(3.16-3.17)E+05 1.66E−03 (1.63-1.68)E−03 5.23 5.1-5.3 PD1B162 3.87E+05(3.84-3.89)E+05 9.79E−04 (9.59-9.98)E−04 2.53 2.5-2.6 PD1B164 2.67E+05(2.67-2.67)E+05 2.87E−04 (2.82-2.91)E−04 1.07 1.06-1.09 PD1B11 2.93E+05(2.85-3.01)E+05 9.17E−03  (0.8-1.00)E−02 31.3 (27.7-35.1) PD1B1833.20E+05 (3.04-3.37)E+05 8.39E−03 (8.01-8.76)E−03 26.3 23.9-28.8 PD1B1842.38E+05 (2.08-2.68)E+05 2.74E−03 (2.55-2.92)E−03 11.5  9.5-14.1 PD1B1853.11E+05 (2.80-3.43)E+05 9.47E−03 (9.38-9.55)E−03 30.5 27.5-34.1 PD1B1872.94E+05 (2.20-3.70)E+05 1.57E−03 (1.28-1.85)E−03 5.32 3.5-8.4 PD1B1923.07E+05 (2.90-3.24)E+05 5.04E−03 (4.86-5.22)E−03 16.4 15.0-18.0

TABLE 11 Antigen: human PD-1 k_(on) k_(off) K_(D) Sample (1/Ms) (1/s)(nM) PD1B70 4.15E+05 4.18E−02 101   PD1B175 4.22E+05 9.72E−03 23  PD1B71 5.48E+05 2.73E−02 49.9 PD1B177 5.15E+05 2.57E−03 5  PD1B1145.17E+05 2.79E−02 54.1 PD1B149 5.32E+05 6.20E−03 ~12*   PD1B160 5.40E+053.71E−03  6.87 PD1B162 6.49E+05 3.86E−03  5.95 PD1B164 4.48E+05 1.31E−03 2.92 PD1B11 5.16E+05 8.52E−03 ~17*   PD1B183 5.27E+05 8.44E−03 16  PD1B184 4.45E+05 5.09E−03 11.4 PD1B185 5.85E+05 7.65E−03 13.1 PD1B1875.35E+05 2.78E−03  5.2 PD1B192 5.41E+05 1.17E−02 ~228    *Values did notpass the data acceptance criteria (chi2 > 20%) and were thereforeconsidered approximations.

Example 4. Combinatorial Variant PD-1 mAb Production

Following the analysis of the affinity results, combinatorial sequenceswere considered.

PD1B11 and PD1B114 have very similar sequences. Because PD1B11 hadapproximately a 3-fold tighter affinity to human PD-1 and a 2-foldtighter affinity to cyno PD-1 compared to PD1B114, antibodies havingcombinations of their various CDRs were made. The HCDR3 of PD1B11 wasplaced into PD1B164 and PD1B162 (affinity-matured variants of PD1B114),using site directed mutagenesis while the HCDR2 of PD1B164 (affinitymatured variant of PD1B114) was placed into PD1B187 (affinity maturedvariant of PD1B11). The resulting heavy chains were paired with parentallight chains resulting in new antibodies PD1B194, PD1B195 and PD1B196,respectively.

PD1B175 and PD1B177 both contained the parental light chain even thoughthe antibodies were generated using diversified VL libraries duringaffinity maturation. In an attempt to increase antibody affinities,PD1B175 heavy chain was paired with PD1L185 or PD1L187 affinity maturedlight chains, and PD1B177 heavy chain was paired with PD1L86, PD1L168 orPD1L190 affinity matured light chains, resulting in antibodies PD1B197,PD1B198, PD1B199, PD1B200 and PD1B201. VH and VL pairing of theantibodies is shown in Table 20 in Example 5.

The HCDR, LCDR, VH and VL sequences of these antibodies are shown inTables 14, 15, 16, 17, 18, 19, 21 and 22 in Example 5. The antibodieswere cloned as IgG2sigma/κ mAbs and transiently expressed in HEK293 expicells for affinity measurements.

Affinities of the resulting antibodies were determined as describedabove. Table 12 shows the measured affinities of the combinatorial mAbvariants to cyno PD-1 and Table 13 shows the affinities to human PD-1.STDEV were calculated for 3 or more replicates generated for human andcyno proteins. If less than 3 replicates were calculated, RANGE isindicated. RANGE is defined as the low and high values for thereplicates tested. For samples without RANGE or STDEV, only oneexperiment was performed

TABLE 12 binding to cyno PD-1 k_(on) STDEV. kon k_(off) STDEV. koffK_(D) STDEV. KD Sample (1/Ms) or RANGE (1/s) or RANGE (nM) or RANGEPD1B70 2.50E+05 (2.25-2.74)E+05 2.22E−02 (2.18-2.26)E−02 88.98(79.6-100)  (Parent) PD1B197 2.75E+05 1.27E+04 1.26E−03 4.04E−05 4.60.3  PD1B198 3.72E+05 1.61E+04 4.16E−03 9.29E−05 11.18 0.54 PD1B113.50E+05 (3.49-3.50)E+05 9.42E−03 (9.38-9.46)E−03 26.95 (26.8-27.1)(Parent) PD1B194 3.22E+05 2.86E+04 1.93E−04 5.86E−06 0.6 0.06 PD1B1954.32E+05 (4.30-4.34)E+05 4.08E−04 (3.96-4.19)E−04 0.94 (0.91-0.97)PD1B196 3.03E+05 6.66E+03 1.76E−04 9.85E−06 0.58 0.03 PD1B71 3.77E+05(3.37-4.17)E+05 1.96E−02 (1.85-2.07)E−02 51.99 (44.4-61.4) (Parent)PD1B199 3.40E+05 7.94E+03 1.77E−04 1.55E−05 0.52 0.05 PD1B200 3.80E+052.21E+04 4.22E−04 1.99E−05 1.11 0.08 PD1B201 3.05E+05 1.80E+04 2.93E−042.35E−05 0.96 0.1 

TABLE 13 binding to human PD-1 k_(on) STDEV. kon k_(off) STDEV. koffK_(D) STDEV. KD Sample (1/Ms) or RANGE (1/s) Or RANGE (nM) or RANGEPD1B70 7.69E+05 (7.37-8.00)E+05 3.49E−02 (3.41-3.56)E−02 45.35(42.6-43.8) (Parent) PD1B197 6.58E+05 2.26E+04 3.24E−03 1.74E−04 4.90.3  PD1B198 8.95E+05 6.44E+04 9.34E−03 9.90E−04 10.43 1.34 PD1B119.33E+05 (8.84-9.82)E+05 9.05E−03 (8.67-9.43)E−03 9.7  (9.6-9.81)(Parent) PD1B194 8.97E+05 1.45E+05 9.60E−04 2.78E−05 1.07 0.18 PD1B1951.23E+06 1.79E+05 1.52E−03 6.51E−05 1.23 0.19 PD1B196 8.83E+05 6.39E+043.66E−04 2.01E−05 0.41 0.04 PD1B71 9.55E+05 (9.33-9.76)E+05 2.25E−02(2.19-2.30)E−02 23.52 (22.4-24.7) (Parent) PD1B199 9.33E+05 6.92E+045.64E−04 1.98E−05 0.6 0.05 PD1B200 1.05E+06 1.40E+05 1.22E−03 3.21E−051.17 0.16 PD1B201 8.58E+05 8.22E+04 9.57E−04 3.06E−05 1.12 0.11

Example 5. Structural Characterization of Anti-PD1 Antibodies Derivedfrom Phage Display Libraries

The cDNA sequences and amino acid translations of the antibodies wereobtained using standard techniques throughout the generation of theantibodies using various campaigns. After polypeptide sequencedetermination, some antibody cDNAs encoding the variable regions or fulllength antibodies were codon optimized using standard methods forscale-up expression.

Table 14 shows the HCDR1 sequences of select PD-1 antibodies.

Table 15 shows the HCDR2 sequences of select PD-1 antibodies.

Table 16 shows the HCDR3 sequences of select PD-1 antibodies.

Table 17 shows the LCDR1 sequences of select PD-1 antibodies.

Table 18 shows the LCDR2 sequences of select PD-1 antibodies.

Table 19 shows the LCDR3 sequences of select PD-1 antibodies.

Table 20 shows the VH and the VL pairing of select PD-1 antibodies.

Table 21 shows the VH sequences of select PD-1 antibodies.

Table 22 shows the VL sequences of select PD-1 antibodies.

TABLE 14 HCDR1 SEQ ID Antibody Sequence NO: PD1B114 S Y A I S 10 PD1B149S Y A I S 10 PD1B160 S Y A I S 10 PD1B162 S Y A I S 10 PD1B164 S Y A I S10 PD1B11 S Y A I S 10 PD1B183 S Y A I S 10 PD1B184 S Y A I S 10 PD1B185S Y A I S 10 PD1B187 S Y A I S 10 PD1B192 S Y A I S 10 PD1B71 S Y A I S10 PD1B177 D Y V I S 11 PD1B70 S Y A I S 10 PD1B175 S Y V I H 12 PD1B194S Y A I S 10 PD1B195 S Y A I S 10 PD1B196 S Y A I S 10 PD1B197 S Y V I H12 PD1B198 S Y V I H 12 PD1B199 D Y V I S 11 PD1B200 D Y V I S 11PD1B201 D Y V I S 11

TABLE 15 HCDR2 SEQ ID Antibody Sequence NO: PD1B114 G I I P I F G T A NY A Q K F Q G 13 PD1B149 G I I P I F G T A N Y A Q K F Q G 13 PD1B160 GI I P I F D T A N Y A Q K F Q G 14 PD1B162 G I I P I F D T A N Y A Q K FQ G 14 PD1B164 G I I P I F D T A N Y A Q K F Q G 14 PD1B11 G I I P I F GT A N Y A Q K F Q G 13 PD1B183 G I I P I F G T A N Y A Q K F Q G 13PD1B184 G I I P I F G T A N Y A Q K F Q G 13 PD1B185 G I I P I F G T A NY A Q K F Q G 13 PD1B187 G I I P I F G T A N Y A Q K F Q G 13 PD1B192 GI I P I F G T A N Y A Q K F Q G 13 PD1B71 G I I P I F G T A N Y A Q K FQ G 13 PD1B177 G I I P I Y G T A N Y A Q K F Q G 15 PD1B70 G I I P I F GT A N Y A Q K F Q G 13 PD1B175 G I I P I F G T A N Y A Q K F Q G 13PD1B194 G I I P I F D T A N Y A Q K F Q G 14 PD1B195 G I I P I F D T A NY A Q K F Q G 14 PD1B196 G I I P I F D T A N Y A Q K F Q G 14 PD1B197 GI I P I F G T A N Y A Q K F Q G 13 PD1B198 G I I P I F G T A N Y A Q K FQ G 13 PD1B199 G I I P I Y G T A N Y A Q K F Q G 15 PD1B200 G I I P I YG T A N Y A Q K F Q G 15 PD1B201 G I I P I Y G T A N Y A Q K F Q G 15

TABLE 16 HCDR3 SEQ ID Antibody Sequence NO: PD1B114 P G L A A A Y D T GN L D Y 16 PD1B149 P G L A A A Y D T G N L D Y 16 PD1B160 P G L A A A YD T G N L D Y 16 PD1B162 P G L A A A Y D T G N L D Y 16 PD1B164 P G L AA A Y D T G N L D Y 16 PD1B11 P G L A A A Y D T G S L D Y 17 PD1B183 P GL A A A Y D T G S L D Y 17 PD1B184 P G L A A A Y D T G S L D Y 17PD1B185 P G L A A A Y D T G S L D Y 17 PD1B187 P G L A A A Y D T G S L DY 17 PD1B192 P G L A A A Y D T G S L D Y 17 PD1B71 G T L D R T G H L D Y18 PD1B177 G T L D R T G H L D Y 18 PD1B70 G Y V R A T G M L D Y 19PD1B175 G Y V R A T G M L D Y 19 PD1B194 P G L A A A Y D T G S L D Y 17PD1B195 P G L A A A Y D T G S L D Y 17 PD1B196 P G L A A A Y D T G S L DY 17 PD1B197 G Y V R A T G M L D Y 19 PD1B198 G Y V R A T G M L D Y 19PD1B199 G T L D R T G H L D Y 18 PD1B200 G T L D R T G H L D Y 18PD1B201 G T L D R T G H L D Y 18

TABLE 17 LCDR1 SEQ ID Antibody Sequence NO: PD1B114 R A S Q S V S S Y LA 20 PD1B149 R A S Q S V R N Y L A 21 PD1B160 R A S Q S V D S Y L A 22PD1B162 R A S Q S V D S Y L A 22 PD1B164 R A S Q S V R S Y L A 23 PD1B11R A S Q S V S S Y L A 20 PD1B183 R A S Q S V S S Y L A 20 PD1B184 R A SQ S V R N Y L A 21 PD1B185 R A S Q S V R N Y L A 21 PD1B187 R A S Q S VR S Y L A 23 PD1B192 R A S Q S V D S Y L A 22 PD1B71 R A S Q S V S S Y LA 20 PD1B177 R A S Q S V S S Y L A 20 PD1B70 R A S Q S V S S Y L A 20PD1B175 R A S Q S V S S Y L A 20 PD1B194 R A S Q S V R S Y L A 23PD1B195 R A S Q S V D S Y L A 22 PD1B196 R A S Q S V R S Y L A 23PD1B197 R A S Q S V S N Y L A 24 PD1B198 R A S Q S V S S Y L A 20PD1B199 R A S Q S V S S Y L A 20 PD1B200 R A S Q S V D N Y L A 25PD1B201 R A S Q S V S N Y L A 24

TABLE 18 LCDR2 SEQ ID Antibody Sequence NO: PD1B114 D A S N R A T 26PD1B149 D A S N R A T 26 PD1B160 D A S D R A T 27 PD1B162 D A S N R A T26 PD1B164 D A S Y R A T 28 PD1B11 D A S N R A T 26 PD1B183 D A S N R AT 26 PD1B184 D A S N R A T 26 PD1B185 D A S D R A T 27 PD1B187 D A S N RA T 26 PD1B192 D A S N R A T 26 PD1B71 D A S N R A T 26 PD1B177 D A S NR A T 26 PD1B70 D A S N R A T 26 PD1B175 D A S N R A T 26 PD1B194 D A SY R A T 28 PD1B195 D A S N R A T 26 PD1B196 D A S N R A T 26 PD1B197 D AS N R A T 26 PD1B198 D A S S R A T 29 PD1B199 D A S T R A T 30 PD1B200 DA S N R A T 26 PD1B201 D A S N R A T 26

TABLE 19 LCDR3 SEQ ID Antibody Sequence NO: PD1B114 Q Q R S N W P L T 31PD1B149 Q Q R N Y W P L T 32 PD1B160 Q Q R G N W P L T 33 PD1B162 Q Q RE Y W P L T 34 PD1B164 Q Q R D Y W P L T 35 PD1B11 Q Q R S N W P L T 31PD1B183 Q Q R G Y W P L T 36 PD1B184 Q Q R N Y W P L T 32 PD1B185 Q Q RW N W P L T 37 PD1B187 Q Q R N Y W P L T 32 PD1B192 Q Q R N Y W P L T 32PD1B71 Q Q R S N W P L T 31 PD1B177 Q Q R S N W P L T 31 PD1B70 Q Q R SN W P L T 31 PD1B175 Q Q R S N W P L T 31 PD1B194 Q Q R D Y W P L T 35PD1B195 Q Q R E Y W P L T 34 PD1B196 Q Q R N Y W P L T 32 PD1B197 Q Q RA Y W P L T 38 PD1B198 Q Q R A E W P L T 39 PD1B199 Q Q R N Y W P L T 32PD1B200 Q Q R S A W P L T 40 PD1B201 Q Q R N Y W P L T 32

TABLE 20 VH VH VL VL peptide SEQ peptide SEQ Antibody ID ID NO: ID IDNO: PD1B114 PD1H24 41 PH9L3 49 PD1B149 PD1H24 41 PD1L128 50 PD1B160PD1H131 42 PD1L101 51 PD1B162 PD1H131 42 PD1L67 52 PD1B164 PD1H131 42PD1L71 53 PD1B11 PD1H3 43 PH9L3 49 PD1B183 PD1H3 43 PD1L109 54 PD1B184PD1H3 43 PD1L128 50 PD1B185 PD1H3 43 PD1L132 55 PD1B187 PD1H3 43 PD1L14856 PD1B192 PD1H3 43 PD1L133 57 PD1B71 PD1H108 44 PH9L3 49 PD1B177PD1H164 45 PH9L3 49 PD1B70 PD1H107 46 PH9L3 49 PD1B175 PD1H163 47 PH9L349 PD1B194 PD1H170 48 PD1L71 53 PD1B195 PD1H170 48 PD1L67 52 PD1B196PD1H170 48 PD1L148 56 PD1B197 PD1H163 47 PD1L185 58 PD1B198 PD1H163 47PD1L187 59 PD1B199 PD1H164 45 PD1L86 60 PD1B200 PD1H164 45 PD1L168 61PD1B201 PD1H164 45 PD1L190 62

TABLE 21 VH VH SEQ peptide ID ID NO: VH sequence PD1H24 41QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGNLDYWGQGT LVTVSS PD1H131 42QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFDTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGNLDYWGQGT LVTVSS PD1H3 43QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGSLDYWGQGT LVTVSS PD1H108 44QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGTLDRTGHLDYWGQGTLVT VSS PD1H164 45QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYVISWVRQAPGQGLEWMGGIIPIYGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGTLDRTGHLDYWGQGTLVT VSS PD1H107 46QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGYVRATGMLDYWGQGTLV TVSS PD1H163 47QVQLVQSGAEVKKPGSSVKVSCKASGGTFKSYVIHWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGYVRATGMLDYWGQGTL VTVSS PD1H170 48QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFDTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGSLDYWGQGT LVTVSS

TABLE 22 VL VL pep-  SEQ tide ID ID NO: VL sequence PH9 49EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPG L3QAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQRSNWPLTFGQGTKVEIK PD150 EIVLTQSPATLSLSPGERATLSCRASQSVRNYLAWYQQKPG L128QAPRLLIHDASNRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQRNYWPLTFGQGTKVEIK PD151 EIVLTQSPATLSLSPGERATLSCRASQSVDSYLAWYQQKPG L101QAPRLLIKDASDRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQRGNWPLTFGQGTKVEIK PD152 EIVLTQSPATLSLSPGERATLSCRASQSVDSYLAWYQQKPG L67QAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQREYWPLTFGQGTKVEIK PD153 EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPG L71QAPRLLIYDASYRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQRDYWPLTFGQGTKVEIK PD154 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPG L109QAPRLLIKDASNRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQRGYWPLTFGQGTKVEIK PD155 EIVLTQSPATLSLSPGERATLSCRASQSVRNYLAWYQQKPG L132QAPRLLIYDASDRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQRWNWPLTFGQGTKVEIK PD156 EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPG L148QAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQRNYWPLTFGQGTKVEIK PD157 EIVLTQSPATLSLSPGERATLSCRASQSVDSYLAWYQQKPG L133QAPRLLIHDASNRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQRNYWPLTFGQGTKVEIK PD158 EIVLTQSPATLSLSPGERATLSCRASQSVSNYLAWYQQKPG L185QAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQRAYWPLTFGQGTKVEIK PD159 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPG L187QAPRLLIEDASSRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQRAEWPLTFGQGTKVEIK PD160 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPG L86QAPRLLIHDASTRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQRNYWPLTFGQGTKVEIK PD161 EIVLTQSPATLSLSPGERATLSCRASQSVDNYLAWYQQKPG L168QAPRLLIHDASNRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQRSAWPLTFGQGTKVEIK PD162 EIVLTQSPATLSLSPGERATLSCRASQSVSNYLAWYQQKPG L190QAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQRNYWPLTFGQGTKVEIK

All anti-PD-1 antibodies were identified to have VH1-69 (SEQ ID NO: 170)and IGKV3-11 (L6) (SEQ ID NO: 171) frameworks.

SEQ ID NO: 170 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAISWVRQAPGQGLEWMG GIIPIFGTANYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCARSEQ ID NO: 171 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP

Example 6. Generation and Characterization of PD-1 Antibodies in Mice

BALB/c were immunized intraperitoneally with huPD1-ECD and assessed forspecific IgG titers. Once sufficient titers were obtained, splenocyteswere isolated and fused with FO cells. The resulting hybridomas wereplated in 96 well plates and cultured for 10 days. Antigen specificclones were identified by standard capture ELISA for binding tohuPD1-ECD. Human PD-1-specific hybridomas were further tested for theiraffinity to human and cyno PD-1, binding to Jurkat cells and cyno PD-L1inhibition. Based on the results, clone PD1B28 was selected forhumanization using framework adaptation.

Framework adaptation process was done as essentially described in U.S.Patent Publ. No. 2009/0118127 and Fransson et al., (2010) J Mol Biol398:214-231. Briefly, the heavy and light chain sequences were comparedwith the human germline sequences (only the “01” alleles as of Oct. 1,2007) using BLAST search against the IMGT database (Kaas, et al., (2004)Nucl Acids Res 32, D208-D210; Lefranc et al., (2005) Nucl Acid Res 33,D593-D597). From this set of human germline genes, redundant genes (100%identical at amino acid level) and those with unpaired cysteine residueswere removed. The remaining closest matching human germline genes inboth the framework and CDR regions were chosen as the acceptor humanframeworks. Several VL and VH germline human frameworks were selectedbased upon overall sequence homology and CDR lengths as well as CDRsimilarity. FR-4 was selected based on sequence similarity of theIGHJ/IGJK germline genes. Then, the CDRs of PD1B28 were transferred intothe selected acceptor human frameworks to generate the HFA variants,except in the region corresponding to the HCDR1 of V_(H). For thisregion a combination of CDR and HV, or a shorter HCDR2 (referred to asKabat-7, see U.S. Patent Publ. No. 2009/0118127) were transferred fromthe non-human antibody into the human FRs because the remaining HCDR2residues have not been found in contact in antigen-antibody complexes ofknown structures (Almagro, (2004) J Mol Recognit 17:132). Backumtationswere introduced into certain residue positions in the humanizedantibodies. PD1B131 backmutations: VH: V37I_Q39L_W47S_R98S, VL: Y49K.PD1B132: VH W47S_R98S, VL: Y49K (residue numbering according toChothia). Select antibodies were expressed as IgG2sigma/κ. The resultingantibodies were characterized for their binding to recombinant PD-1 andPD-1 expressed on cells (Jurkat cells), and their ligand inhibition(cyno PD-L1 and human PD-L1). Characteristics of select humanizedantibodies are shown in Table 23. The VH and the VL sequences of thegenerated antibodies are shown in Table 24 and Table 25, respectively.

TABLE 23 Jurkat cell PD-L1 Inhibition, binding Human PD-1 Affinity IC₅₀(ng/ml) relative to kon koff K_(D) Human Cyno mAb PD1B28 (1/Ms) (1/s)(pM) PD-L1 PD-L1 PD1B28 100% 9.70E+05 1.18E−04 122 67 96 PD1B131 100%8.27E+05 1.05E−04 127 79 96 PD1B132 100% 9.14E+05 8.80E−05 96 55 79

TABLE 24 VH VL VH SEQ mAb ID ID VH sequence ID NO: PD1B131 PD1H130PD1L62 EVQLVESGGGLVQPGG 63 SLRLSCAASGFAFSRY DMSWIRLAPGKGLESVAYISGGGANTYYLDNV KGRFTISRDNAKNSLY LQMNSLRAEDTAVYYC ASPYLSYFDVWGQGTLVTVSS PD1B132 PD1H129 PD1L62 EVQLVESGGGLVQPGG 64 SLRLSCAASGFAFSRYDMSWVRQAPGKGLESV AYISGGGANTYYLDNV KGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCASPYLSYFDVWGQGTL VTVSS

TABLE 25 VH VL VL SEQ mAb ID ID VL sequence ID NO: PD1B131 PD1H130PD1L62 EIVMTQSPATLSVSPG 65 ERATLSCRASQSLSDY LHWYQQKPGQAPRLLIKSASQSISGIPARFSG SGSGTEFTLTISSLQS EDFAVYYCQNGHSFPY TFGQGTKLEIK PD1B132PD1H129 PD1L62 EIVMTQSPATLSVSPG 65 ERATLSCRASQSLSDY LHWYQQKPGQAPRLLIKSASQSISGIPARFSG SGSGTEFTLTISSLQS EDFAVYYCQNGHSFPY TFGQGTKLEIKThe CDR sequences of PD1B131 and PD1B132 are shown below:

HCDR1 (SEQ ID NO: 66) RYDMS HCDR2 (SEQ ID NO: 67) YISGGGANTYYLDNVKGHCDR3 (SEQ ID NO: 68) PYLSYFDV LCDR1 (SEQ ID NO: 69) RASQSLSDYLH LCDR2(SEQ ID NO: 70) SASQSIS LCDR3 (SEQ ID NO: 71) QNGHSFPYT

Example 7. Effect of Isotype Switching on Anti-PD-1 Antibody Properties

Variable regions of antibodies PD1B196 and PD1B199 (of IgG2sigma/κisotype) were cloned as IgG4 S228P isotypes and variable regions fromantibody PD1B132 (of IgG2) into IgG2sigma isotype to assess possibledifferences in functionality and developability.

The antibodies were named PD1B244 (PD1B196 VH/VL on IgG4 S228P) PD1B245(PD1B199 VH/VL on IgG4 S228P) AND PD1B243 (PD1B132 VH/VL on IgG2sigma).

Isotype switch had no consistent effect on the antibody propertieshowever, for some of the antibodies, some change in EC₅₀ values wereseen in the CMV assay.

Exemplified below are heavy chain and light chain amino acid sequencesof various antibodies. Table 26 shows the summary of the VH, VL, heavychain and light chain SEQ ID NOs: for select antibodies.

TABLE 26 VH VH VL VL HC LC peptide SEQ peptide SEQ SEQ SEQ Antibody IDID NO: ID ID NO: ID NO ID NO: PD1B114 PD1H24 41 PH9L3 49 212 213 PD1B149PD1H24 41 PD1L128 50 214 215 PD1B160 PD1H131 42 PD1L101 51 216 217PD1B162 PD1H131 42 PD1L67 52 218 219 PD1B164 PD1H131 42 PD1L71 53 220221 PD1B183 PD1H3 43 PD1L109 54 222 223 PD1B184 PD1H3 43 PD1L128 50 224225 PD1B185 PD1H3 43 PD1L132 55 226 227 PD1B192 PD1H3 43 PD1L133 57 228229 PD1B243 PD1H129 64 PD1L62 65 74 75 PD1B244 PD1H170 48 PD1L148 56 7273 PD1B245 PD1H164 45 PD1L86 60 76 77

HC of PD1B244 SEQ ID NO: 72QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFDTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGSLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK LC of PD1B244 SEQ ID NO: 73EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNYWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC of PD1B243 SEQ ID NO: 74EVQLVESGGGLVQPGGSLRLSCAASGFAFSRYDMSWVRQAPGKGLESVAYISGGGANTYYLDNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLSYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGKLC of PD1B243 SEQ ID NO: 75EIVMTQSPATLSVSPGERATLSCRASQSLSDYLHWYQQKPGQAPRLLIKSASQSISGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQNGHSFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC of PD1B245 SEQ ID NO: 76QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYVISWVRQAPGQGLEWMGGIIPIYGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGTLDRTGHLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGKLC of PD1B245 SEQ ID NO: 77EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIHDASTRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNYWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC of PD1B114 SEQ ID NO: 212QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGNLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LC of PD1B114 SEQ ID NO: 213EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC of PD1B149 SEQ ID NO 214QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGNLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LC of PD1B149 SEQ ID NO: 215EIVLTQSPATLSLSPGERATLSCRASQSVRNYLAWYQQKPGQAPRLLIHDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNYWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC of PD1B160 SEQ ID NO: 216QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFDTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGNLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LC of PD1B160 SEQ ID NO: 217EIVLTQSPATLSLSPGERATLSCRASQSVDSYLAWYQQKPGQAPRLLIKDASDRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRGNWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC of PD1B162 SEQ ID NO: 218QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFDTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGNLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LC of PD1B162 SEQ ID NO: 219EIVLTQSPATLSLSPGERATLSCRASQSVDSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQREYWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC of PD1B164 SEQ ID NO: 220QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFDTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGNLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LC of PD1B164 SEQ ID NO: 221EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASYRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRDYWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC of PD1B183 SEQ ID NO: 222QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGSLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LC of PD1B183 SEQ ID NO: 223EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIKDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRGYWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC of PD1B184 SEQ ID NO: 224QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGSLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LC of PD1B184 SEQ ID NO: 225EIVLTQSPATLSLSPGERATLSCRASQSVRNYLAWYQQKPGQAPRLLIHDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNYWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC of PD1B185 SEQ ID NO: 226QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGSLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LC of PD1B185 SEQ ID NO: 227EIVLTQSPATLSLSPGERATLSCRASQSVRNYLAWYQQKPGQAPRLLIYDASDRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRWNWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC of PD1B192 SEQ ID NO: 228QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGSLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LC or PD1B192 SEQ ID NO: 229EIVLTQSPATLSLSPGERATLSCRASQSVDSYLAWYQQKPGQAPRLLIHDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNYWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Example 8. Characterization of PD-1 Antibodies in Cell-Based Assays

Select antibodies were characterized in MLR and CMV assays usingprotocols described in Example 1. The EC₅₀ values for IFN-γ inductionfrom MLR and CMV assays are shown in Table 27. In most cases, anti-PD-1antibodies showed a dose-dependent increase in IFN-γ levels in both MLRand CMV assays.

TABLE 27 MLR CMV Origin mAb EC₅₀, nM EC₅₀, nM Phage PD1B3 0.29 0.06display PD1B91 0.05 0.03 PD1B194 NT NC PD1B195 NT 1.64 PD1B196 0.14 0.31PD1B199 0.63 NC PD1B200 NT 3.81 PD1B201 NT 2.60 PD1B244 0.08 0.03 HFAPD1B132 NT 0.07 PD1B243 0.07 0.02 NT: not tested NC: no convergence HFA:human framework adaptation

In addition to IFN-γ, secreted levels of additional cytokines were alsoaffected by PD-1 blockade in the two assays. Upon CMV stimulation,anti-PD-1 antibodies led to a dose-dependent induction of TNF-α andIL-4, whereas in the MLR assay they increased TNF-α and IL-2 levels.

Example 9. Generation of Human Anti-TIM-3 Antibodies Using Phage DisplayLibraries

The de novo pIX Fab libraries described in Example 2 were panned againstthe extracellular domain of recombinant human TIM-3-Fc fusion protein(R&D Systems, #2365-TM; residues Ser22-Arg200 of full length TIM-3)(huTIM-3-Fc).

The recombinant protein was biotinylated (bt) and captured onstreptavidin magnetic beads (Dynal), then exposed to the de novo pIX Fablibraries at a final concentration of 100 nM. Non-specific phages werewashed away in PBS-Tween and bound phages were recovered by infection ofMC1061F′ E. coli cells. Phages were amplified from these cells overnightand panning was repeated for a total of three rounds. Following thefinal round of biopanning, monoclonal Fab was screened for binding tobiotinylated human TIM-3-Fc captured on ELISA plates by Streptavidin andsecreted Fab was added to the captured antigen, followed by detection ofthe Fab with Goat Anti human kappa:HRP. Select antibodies were expressedand cloned on various IgG isotypes as indicated below, and characterizedfurther.

Example 10. Generation of Anti-TIM-3 Antibodies in Mice

Balb/c mice were immunized with recombinant human TIM-3-Fc fusionprotein (R&D Systems, catalog #2365-TM) over the course of 18 days.Spleens were harvested, and a B cell enriched population was fused withFO mouse myeloma cells to generate mAb secreting hybridomas. Thehybridoma supernatants were screened for binding by ELISA to TIM-3-Fcprotein and an irrelevant human IgG1 Fc. TIM-3 specific supernatantswere then assayed for the ability to bind to TIM-3 expressing THP-1cells.

Select mAb HC and LC v-genes were cloned from the TIM-3 positivehybridomas using standard molecular biology techniques (RT-PCR followedby PCR fragment ligation into plasmid expression vectors). mAbs wereexpressed recombinantly, and the ELISA was repeated to confirm TIM-3specific binding. Molecular models for murine antibody sequences to behuman framework adapted were constructed using MOE (CCG, Montreal) andvisually inspected. Potential problem positions that might influenceantigen binding, VL/VH packing and/or core residues that might affectdomain stabilities were identified. For both VL and VH, multiple humanframeworks were proposed with or without back mutations to mouseframework sequences if problem positions were identified. The designedsequences were cloned into heavy and light chain plasmids and expressedin Expi293F cells. Expressed antibody in the culture supernatants werequantified and assessed for binding to HEK293 cells transfected withrecombinant human TIM-3.

Example 11. Isotypes of Anti-TIM-3 Antibodies

The VH and VL of isolated anti-TIM-3 antibodies were cloned onto variousheavy chain isotypes, optionally with various Fc substitutions, andallotypes with κ light chains during the course of antibodycharacterization to evaluate the effect, if any, of isotype switch onfunctionality or developability of the antibodies. The various isotypesused are shown in Table 28.

TABLE 28 Substitution when compared to wild Purpose of Isotype type*substitution IgG2sigma V234A, G237A, Abolishing effector P238S, H268A,functions V309L, A330S, P331S IgG2sigma_K409R V234A, G237A, Abolishingeffector P238S, H268A, functions, improving V309L, A330S, heterodimerformation P331S, K409R in bispecific antibody IgG2sigma_F405L V234A,G237A, Abolishing effector P238S, H268A, functions, improving V309L,A330S, heterodimer formation P331S, F405L in bispecific antibodyIgG4_PAA S228P, F234A, Antibody stability, L235A abolishing effectorfunctions IgG4_PAA_F405L_R409K S228P, F234A, Antibody stability, L235A,F450L, abolishing effector R409K functions, improving heterodimerformation in bispecific antibody IgG4_S228P S228P Antibody stabilityIgG1 Wild type IgG1sigma L234A, L235A, Abolishing effector G237A, P238S,functions H268A, A330S, P331S IgG1sigma_K409R L234A, L235A, Abolishingeffector G237A, P238S, functions, improving H268A, A330S, heterodimerformation P331S, K409R in bispecific antibody IgG1sigma_F405L L234A,L235A, Abolishing effector G237A, P238S, functions, improving H268A,A330S, heterodimer formation P331S, F405L in bispecific antibody IgG1_AAL234A, L235A Abolishing effector functions *Residue numbering accordingto the EU IndexThe various allotypes used in the generated antibodies are shown inTable 29. Some of the antibodies had chimeric allotypes. AntibodiesTM3B105 and TM3B403 for example differ by one amino acid substitution ina constant region at position 189. TM3B105 heavy and light chains SEQ IDNOs: 240 and 79, respectively; TM3B403 heavy and light chains SEQ IDNOs: 78 and 79, respectively. The two antibodies are expected to havethe same characteristics.

TABLE 29 Isotype/Allotype/Substitutions IgG2sigma_G2m(n−)/(n)_K409RIgG2sigma_G2m(n−)_K409R IgG2sigma_G2m(n−)/(n) IgG2sigma_F405L IgG2_K409RIgG2sigma_G2m(n−) IgG2 IgG4_S228P IgG4_S228P_F405L_R409KIgG4_nG4m(a)_PAA_F405L_R409K IgG4_PAA IgG1sigma IgG1_G1m(17)IgG1_G1m(17,1)_AA

In general, anti-TIM-3 antibodies with IgG2sigma Fc had greater activityin the CMV assay than anti-TIM-3 antibodies with huIgG4 Fc. In addition,antibodies with huIgG2 Fc demonstrated functionality that wasintermediate between IgG2sigma and IgG4. Allotype had no effect onantibody activity.

Example 12. Structural Characterization of Anti-TIM-3 Antibodies

The cDNA sequences and amino acid translations of the antibodies wereobtained using standard techniques throughout the generation of theantibodies using various campaigns. After polypeptide sequencedetermination, some antibody cDNAs encoding the variable regions or fulllength antibodies were codon optimized using standard methods forscale-up expression. Antibodies TM3B103, TM3B105, M3B108, TM3B109 andTM3B113 were isolated from phage display libraries. Antibodies TM3B189,TM3B190, TM3B193, TM3B195 and TM3B196 were generated by immunizing mice.

Table 30 shows the HCDR1 sequences of select anti-TIM-3 antibodies.

Table 31 shows the HCDR2 sequences of select anti-TIM-3 antibodies.

Table 32 shows the HCDR3 sequences of select anti-TIM-3 antibodies.

Table 33 shows the LCDR1 sequences of select anti-TIM-3 antibodies.

Table 34 shows the LCDR2 sequences of select anti-TIM-3 antibodies.

Table 35 shows the LCDR3 sequences of select anti-TIM-3 antibodies.

Table 36 shows the VH sequences of select anti-TIM-3 antibodies.

Table 37 shows the VL sequences of select anti-TIM-3 antibodies.

Table 38 shows the frameworks of select anti-TIM-3 antibodies.

TABLE 30 HCDR1 mAb SEQ name Sequence ID NO: TM3B103 N Y W M S 90 TM3B105S Y A M S 91 TM3B109 S Y A M S 91 TM3B108 G Y W M H 92 TM3B113 D Y W M S93 TM3B189 S Y V M Y 94 TM3B190 S D Y A W N 95 TM3B193 D T Y L H 96TM3B195 S Y W M Q 97 TM3B196 S Y G V H 98 TM3B291 S Y W M Q 97

TABLE 31 HCDR2 SEQ ID mAb Sequence NO: TM3B103 A I S G S G G S T Y Y A DS V K G  99 TM3B105 A I S G S G G S T Y Y A D S V K G  99 TM3B109 A I SG S G G S T Y Y A D S V K G  99 TM3B108 A I S Y S G S S T Y Y A D S V KG 100 TM3B113 V I K Y S G G S K Y Y A D S V K G 101 TM3B189 Y I N P Y ND G T K Y N E K F K G 102 TM3B190 Y I N Y S G R T S Y N P S L K S 103TM3B193 R I D P T N G N I K Y D P K F Q G 104 TM3B195 A I Y P G D G D IR Y T Q N F K G 105 TM3B196 V I W S D G S T T Y N S A L K S 106 TM3B291A I Y P G D G D I R Y T Q N F K G 105

TABLE 32 HCDR3 SEQ  ID mAb Sequence NO: TM3B103 D H W D P N F L D Y 107TM3B105 S P Y A P L D Y 108 TM3B109 N E E P D D R L D Y 109 TM3B108 G TN W L D Y 110 TM3B113 E L E G V F D Y 111 TM3B189 D D Y D V A P F A Y112 TM3B190 G G N F D Y 113 TM3B193 P Y Y G F F D Y 114 TM3B195 W E K ST T V V Q R N Y F D Y 115 TM3B196 Q A N Y R Y D S A M D Y 116 TM3B291 WE K S T T V V Q R N Y F D Y 115

TABLE 33 LCDR1 SEQ ID mAb Sequence NO: TM3B103 R A S Q S V S S S Y L A117 TM3B105 R A S Q S V N D Y L A 118 TM3B109 K S S Q S V L A S S N N KN Y L A 119 TM3B108 R A S Q S V S S S Y L A 117 TM3B113 R A S Q S V S NS T L A 120 TM3B189 R A S E S L D S Y G N S Y I H 121 TM3B190 Q A T Q DI V K N L N 122 TM3B193 K A S Q D V N T A V A 123 TM3B195 K A S E N V GT F V S 124 TM3B196 K A S Q S V D Y D G D S Y M N 125 TM3B291 K A S E NV G T F V S 124

TABLE 34 LCDR2 SEQ ID mAb Sequence NO: TM3B103 G A S S R A T 126 TM3B105D A S N R A T 127 TM3B109 W A S T R E S 128 TM3B108 G A S S R A T 126TM3B113 T A S S R A T 129 TM3B189 L A S N L E S 130 TM3B190 Y V T E L AE 131 TM3B193 S A T Y R Y T 132 TM3B195 G A S N R Y T 133 TM3B196 T A AN L Q S 134 TM3B291 G A S N R Y T 133

TABLE 35 LCDR3 SEQ ID mAb Sequence NO: TM3B103 Q Q Y G S S P L T 135TM3B105 Q Q G G H A P I T 136 TM3B109 Q Q Y Y S T P L T 137 TM3B108 Q QY G S S P L T 135 TM3B113 Q Q S Y T S P W T 139 TM3B189 Q Q N N E D P FT 140 TM3B190 L Q F Y E F P L T 141 TM3B193 Q Q H Y S T P Y T 142TM3B195 G Q S Y S Y P T 143 TM3B196 Q Q S N E D P F T 144 TM3B291 G Q SY S Y P T 143

TABLE 36 mAb VH SEQ name name VH sequence ID NO: TM3B103 TM3H21EVQLLESGGGLVQPGGSLRLS 145 CAASGFTFSNYWMSWVRQAPG KGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCAKDHWDPNF LDYWGQGTLVTVSS TM3B105TM3H24 EVQLLESGGGLVQPGGSLRLS 146 CAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADS VKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCAKSPYAPLDYWGQGTLVTVSS TM3B108 TM3H30 EVQLLESGGGLVQPGGSLRLS 147CAASGFTFSGYWMHWVRQAPG KGLEWVSAISYSGSSTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTNWLDY WGQGTLVTVSS TM3B109 TM3H31 EVQLLESGGGLVQPGGSLRLS148 CAASGFTFSSYAMSWVRQAPG KGLEWVSAISGSGGSTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNEEPDDR LDYWGQGTLVTVSS TM3B113 TM3H65EVQLLESGGGLVQPGGSLRLS 149 CAASGFTFSDYWMSWVRQAPG KGLEWVSVIKYSGGSKYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCAKELEGVFD YWGQGTLVTVSS TM3B189 TM3H141EVQLQQSGPELLKPGASVKMS 150 CKASGYTFTSYVMYWVKQKPG QGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELS RLTSEDSAVYYCTRDDYDVAP FAYWGQGTLVTVSA TM3B190TM3H96 DVQLQESGPGLVKPSQSLSLT 151 CTVTGYSITSDYAWNWIRQFPGNKLEWMGYINYSGRTSYNPS LKSRISITRDTSKNQFFLQLN SVTTEDTATYYCTSGGNFDYWGQGTTLTVSS TM3B193 TM3H99 EVQLQQSGAELVKPGASVKLS 152CTASGFHIKDTYLHWVKQRPE QGLEWIGRIDPTNGNIKYDPK FQGKATITSDTSSNTAYLQLSSLTSEDTAVYYCARPYYGFFD YWGQGTTLTVSS TM3B195 TM3H144 EVQLQQSGAELARPGASVKLS153 CKASGYTFTSYWMQWVKQRPG QGLEWIGAIYPGDGDIRYTQN FKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARWEKSTTV VQRNYFDYWGQGTTLTVSS CORRECT? TM3B196 TM3H102QVQLKESGPGLVAPSQSLSIT 154 CTISGFSLTSYGVHWVRQPPG KGLEWLVVIWSDGSTTYNSALKSRLSISKDNSKSQVFLKMNS LQTDDTAMYYCARQANYRYDS AMDYWGQGTSVTVSS TM3B291TM3H162 EVQLVQSGAEVKKPGESLKIS 172 CKGSGYSFTSYWMQWVRQMPGKGLEWMGAIYPGDGDIRYTQN FKGQVTISADKSISTAYLQWS SLKASDTAMYYCARWEKSTTVVQRNYFDYWGQGTTVTVSS

TABLE 37 mAb VL SEQ name name VL sequence ID NO: TM3B103 PH9L1EIVLTQSPGTLSLSPGERATL 155 SCRASQSVSSSYLAWYQQKPG QAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQYGSSPLTFGQGTKV EIK TM3B105 TM3L33EIVLTQSPATLSLSPGERATL 156 SCRASQSVNDYLAWYQQKPGQ APRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFA VYYCQQGGHAPITFGQGTKVE IK TM3B108 PH9L1EIVLTQSPGTLSLSPGERATL 155 SCRASQSVSSSYLAWYQQKPG QAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQYGSSPLTFGQGTKV EIK TM3B109 PYYL6DIVMTQSPDSLAVSLGERATI 157 NCKSSQSVLASSNNKNYLAWY QQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSL QAEDVAVYYCQQYYSTPLTFG QGTKVEIK TM3B113 TM3L12EIVLTQSPGTLSLSPGERATL 158 SCRASQSVSNSTLAWYQQKPG QAPRLLIYTASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQSYTSPWTFGQGTKV EIK TM3B189 TM3L61DIVLTQSPASLAVSLGQRATI 159 SCRASESLDSYGNSYIHWYQQ KPGQPPKLLIYLASNLESGVPARFSGSGSKTDFTLTIDPVEA DDPATYYCQQNNEDPFTFGSG TKLEIK TM3B190 TM3L62DIVMTQSPSSMSASLGDRITI 160 TCQATQDIVKNLNWYQQKPGK PPSFLIHYVTELAEGVPSRFSGSGSGSDYSLTISNLESEDFA DYYCLQFYEFPLTFGAGTKLE LK TM3B193 TM3L52DIVMTQSHKFMSTSVGDRVSI 161 TCKASQDVNTAVAWYQQKPGQ SPKLLIYSATYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLA VYYCQQHYSTPYTFGSGTKLE IK TM3B195 TM3L67DVQMIQSPKSMSMSVGERVTL 162 SCKASENVGTFVSWYQQKPDQ SPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLA DYHCGQSYSYPTFGSGTKLEM TM3B196 TM3L64DIQMTQSPASLAVSLGQRATI 163 SCKASQSVDYDGDSYMNWYQQ KPGQPPKLLIYTAANLQSGIPARFSGSGSGTDFTLNIHPVEE EDAATYYCQQSNEDPFTFGSG TKLEIK TM3B291 TM3L85DIQMTQSPSSLSASVGDRVTI 173 TCKASENVGTFVSWYQQKPGK APKLLIYGASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCGQSYSYPTFGQGTKLEI K

TABLE 38 VH framework VL framework SEQ SEQ mAb VH ID VL ID name nameName NO: name Name NO: TM3B103 TM3H21 IGHV3-23 174 PH9L1 IGKV3-20 180TM3B105 TM3H24 IGHV3-23 174 TM3L33 IGKV3-11 171 TM3B108 TM3H30 IGHV3-23174 PH9L1 IGKV3-20 180 TM3B109 TM3H31 IGHV3-23 174 PYYL6 IGKV4-1 181TM3B113 TM3H65 IGHV3-23 174 TM3L12 IGKV3-20 180 TM3B189 TM3H141 IGHV1-02175 TM3L61 IGKV4-1 181 TM3B190 TM3H96 IGHV4-30 176 TM3L62 IGKV1-39 182TM3B193 TM3H99 IGHV1-03 177 TM3L52 IGKV1-33 183 TM3B195 TM3H144 IGHV1-03177 TM3L67 IGKV1-39 182 TM3B196 TM3H102 IGHV2-26 178 TM3L64 IGKV4-1 181TMB291 TM3H162 IGHV5-51 179 TM3L85 IGKV1-39 182

IGHV3-23 SEQ ID NO: 174EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK IGHV1-02SEQ ID NO: 175 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGRINPNSGGTNYAQKFQGRVTSTRDTSISTAYMELSRLRSDDTVVYYCAR IGHV4-30SEQ ID NO: 176 QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA R IGHV1-03SEQ ID NO: 177 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR IGHV2-26SEQ ID NO: 178 QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCA RI IGHV5-51SEQ ID NO: 179 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGHYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCAR IGKV3-20 SEQ ID NO: 180EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP IGKV3-11 SEQ ID NO: 171EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP IGKV4-1 SEQ ID NO: 181EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP IGKV1-39 SEQ ID NO: 182DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPI GKV1-33 SEQ ID NO: 183DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLP

Example 13. Characterization of Anti-TIM-3 Antibodies

Select antibodies were characterized for their binding to human or cynocells, and their ability to block ligand galectin 9 binding. Table 39shows the characteristics of select antibodies in these assays. The cellbinding data represents the calculated EC₅₀ values of the antibodiesbinding to cells transfected with the indicated TIM-3 recombinantprotein expressed in μg/ml units. The galectin-9 inhibition representsthe maximal level of inhibition of galectin-9 binding to human TIM-3seen with the indicated antibodies. The tested antibodies were tested asIgG2sigma isotypes.

Epitope mapping assays were performed by coating recombinant huTIM-3-Fcprotein on MSD plates. Plates were blocked and washed, followed by theaddition of the mixture of the MSD-tag-labeled anti-TIM-3 mAbs incubatedwith increasing concentrations of unlabeled anti-TIM-3 mABs. Afterincubation with gentle shaking at room temperature, plates were washedand analyzed with a SECTOR Imager 6000. Antibodies that competed witheach other for binding to human TIM-3 were considered to bind to similarepitopes. Positive inhibition was noted if >75% of the binding wasinhibited. Partial inhibition was 40-75% inhibition. <40% inhibition wasdenoted as negative.

TABLE 39 Cell binding EC₅₀, μg/ml Galectin 9 Human Cyno Inhibition,Epitope mAb cells cells % inhibition Bin TM3B103 0.71 0.09 71.2 1TM3B105 0.46 0.03 69.8 1 TM3B107 74.8 2 TM3B108 0.42 0.03 64.2 1 TM3B10977.0 1 TM3B113 75.6 2 TM3B189 0.74 0.19 76.4 3 TM3B190 0.35 0.08 60.7 1TM3B193 47.4 3 TM3B219 0.60 0.10 38.0 3 TM3B196 57.0 4

Example 14. Development of a Functional In Vitro Assay to CharacterizeAnti-TIM-3 Antibodies

Functional assessment of inhibitory receptors such as PD-1 can be doneusing T cells from normal donor that are stimulated by allogeneicdendritic cells or specific antigens, such as Tetanus toxoid or CMV. Inthis setting, changes in T cell function with antibody treatment can bedetected by measuring supernatant cytokine levels or markers of T cellactivation. Effects of anti-TIM-3 antibodies can be very variable inthese types of assays, with little overall change in the state ofactivation or functionality of bulk T cell (non-antigen-specific). Onthe other hand, using tetramer approaches to follow single T cellsub-populations/clones in these assays does not provide the resolutionneeded to detect functional effects of anti-TIM-3 antibodies, due to thelow frequency and heterogeneous functional profile of these T cellclones. In addition, this approach necessitates the prior identificationof the epitopes recognized by CMV-specific T cells in each donor.

CD137 was recently described as a surrogate marker for activatedantigen-specific T cells (Wolf et al., (2007) Blood 110(1):201-210;Klinger et al., (2013) PLoS One 8(9): e74231). In our assays, usingCD137 enabled the identification of antigen specific CD8⁺ and CD4⁺ Tcells that expand in response to CMV antigen stimulation and allowed thedetection of the functional effects of anti-TIM-3 antibodies. Inaddition to CD137 expression, cytokine secretion by MSD was alsoevaluated in these assays.

The activity of select anti-TIM-3 antibodies was tested in CMVpp65-stimulated PBMCs. In these assays, anti-TIM-3 antibodies augmentedT cell activation, as evidenced by increased CD137 expression on bothCD8⁺ and CD4⁺ T cells. In addition, selected anti-TIM-3 antibodies alsoenhanced secretion of IFN-γ and TNF-α in this assay.

Table 40 shows the results of the CMV assay where enhanced surfaceexpression of CD137 was evaluated on CD8+ or CD4+ cells for select TIM-3antibodies. The table shows the p values generated using the Two-tailedT-test (unequal variance).

TABLE 40 CD8⁺CD137⁺, p values CD4⁺CD137⁺, p values Mean Std Dev n MeanStd Dev n TM3B103 0.043 0.025 5 0.071 0.112 3 TM3B105 0.029 0.036 6 0.010.017 3 TM3B107 0.182 0.188 5 0.157 0.125 3 TM3B108 0.022 0.018 5 0.010.01  3 TM3B109 0.035 0.041 5 0.017 0.015 3 TM3B113 0.082 0.064 6 0.050.026 3 TM3B189 0.027 0.026 6 0.007 0.011 3 TM3B190 0.078 0.159 6 0.0040.005 3 TM3B193 0.467 0.252 3 0.1 NA 1 TM3B195 0.035 0.043 7 0.01 0.01 3 TM3B196 0.328 0.183 6 0.733 0.058 3 TM3B197 0.473 0.303 4 0.3 NA 1

Example 15. Generation of Bispecific PD-1/TIM-3 Antibodies

Select monospecific PD-1 and TIM-3 antibodies were expressed as IgG1/κ,IgG2/κ or IgG4/κ. Substitutions were made at positions 405 and 409 (EUnumbering) in the monospecific antibodies to promote subsequent in vitroarm exchange and formation of the bispecific antibodies. The IgG1 andIgG2 anti-PD-1 and anti-TIM-3 antibodies were engineered to have a F405Land a K409R substitution, respectively, to promote arm exchange andgeneration the bispecific antibodies. On IgG4, the 409 WT position is R,hence the IgG4 anti-PD-1 antibody was not engineered and the IgG4anti-TIM-3 antibody was engineered to have F405L and R409Ksubstitutions. In addition to position 405 and 409 substitutions, theIgG4 mAbs were engineered to have S228P substitution and the IgG2antibodies were optionally engineered to include IgG2sigma substitution(V234A, G237A, P238S, H268A, V309L, A330S and P331S).

The monospecific antibodies were expressed and purified using standardmethods using a Protein A column (HiTrap Mab Select SuRe column) Afterelution, the pools were dialyzed into D-PBS, pH 7.2

Bispecific PD-1/TIM-3 antibodies were generated by combining amonospecific PD-1 mAb and a monospecific TIM-3 mAb in in vitro Fab armexchange as described in Int. Patent Publ. No. WO2011/131746. Briefly,at about 1-20 mg/ml at a molar ratio of 1:1 of each antibody in PBS, pH7-7.4 and 75 mM 2-mercaptoethanolamine (2-MEA) was mixed together andincubated at 25-37° C. for 2-6 h, followed by removal of the 2-MEA viadialysis, diafiltration, tangential flow filtration and/or spinned cellfiltration using standard methods.

The bispecific antibodies were further purified after the in vitroFab-arm exchange using hydrophobic interaction chromatography tominimize residual parental PD-1 and TIM-3 antibodies using standardmethods.

Select monospecific anti-PD-1 antibodies and anti-TIM-3 antibodies werecombined in matrix in in vitro Fab arm exchange to generate bispecificantibodies. Table 41, Table 42 and Table 43 show the VH, the VL, the HCand the LC sequences of the generated bispecific antibodies and theirisotypes. The G2 antibody allotypes were G2m(n)/(n−) or G2m(n−).

In some experiments, control antibodies were used that were monovalentfor either PD-1 or TIM-3 with the second arm being inert binding togp120. The gp120 binding arm had a VH of SEQ ID NO: 184 and the VL ofSEQ ID NO: 185. Table 44 shows the generated control antibodies.

VH of gp120 binding mAb SEQ ID NO: 184QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHWVRQAPGQRFEWMGWINPYNGNKEFSAKFQDRVTFTADTSANTAYMELRSLRSADTAVYYCARVGPYSWDDSPQDNYYMDVWGKGTTVIVSS VL of gp120 binding mAb SEQ ID NO: 185EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFTLTITRVEPEDFALYYCQVYGASSYT FGQGTKLERK

TABLE 41 PD-1 binding arm VH1 VL1 SEQ SEQ mAb VH1 ID NO: VL1 ID NO:Isotype PTBB14 PD1H170 48 PD1L148 56 IgG2sigma PTBB15 PD1H170 48 PD1L14856 IgG2sigma PTBB16 PD1H129 64 PD1L62 65 IgG2sigma PTBB17 PD1H129 64PD1L62 65 IgG2sigma PTBB24 PD1H170 48 PD1L148 56 IgG2sigma PTBB30PD1H170 48 PD1L148 56 IgG2sigma PTBB27 PD1H170 48 PD1L148 56 IgG2 PTBB28PD1H170 48 PD1L148 56 IgG2 PTBB18 PD1H129 64 PD1L62 65 IgG4_S228P PTBB20PD1H170 48 PD1L148 56 IgG4_S228P PTBB21 PD1H170 48 PD1L148 56 IgG4_S228P

TABLE 42 TIM-3 binding arm VH2 VL2 SEQ SEQ mAb VH2 ID NO: VL2 ID NO:Isotype PTBB14 TM3H144 153 TM3L67 162 IgG2sigma PTBB15 TM3H24 146 TM3L33156 IgG2sigma PTBB16 TM3H144 153 TM3L67 162 IgG2sigma PTBB17 TM3H24 146TM3L33 156 IgG2sigma PTBB24 TM3H162 172 TM3L85 173 IgG2sigma PTBB30TM3H24 146 TM3L33 156 IgG2sigma PTBB27 TM3H162 172 TM3L85 173 IgG2PTBB28 TM3H24 146 TM3L33 156 IgG2 PTBB18 TM3H24 146 TM3L33 156 IgG4_S228PTBB20 TM3H24 146 TM3L33 156 IgG4_S228 PTBB21 TM3H162 172 TM3L85 173IgG4_S228

TABLE 43 SEQ ID NO: PD-1 binding arm TIM-3 binding arm mAb HC1 LC1 HC2LC2 PTBB14 186 188 190 193 PTBB15 186 188 191 194 PTBB16 187 189 190 193PTBB17 187 189 191 194 PTBB24 186 188 192 195 PTBB30 186 188 248 194PTBB27 241 188 244 195 PTBB28 241 188 245 194 PTBB18 242 189 246 194PTBB20 243 188 246 194 PTBB21 243 188 247 195

SEQ ID NO: 186 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFDTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGSLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKSEQ ID NO: 187 EVQLVESGGGLVQPGGSLRLSCAASGFAFSRYDMSWVRQAPGKGLESVAYISGGGANTYYLDNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLSYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK SEQ ID NO: 188EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNYWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO: 189 EIVMTQSPATLSVSPGERATLSCRASQSLSDYLHWYQQKPGQAPRLLIKSASQSISGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQNGHSFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO: 190 EVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGAIYPGDGDIRYTQNFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARWEKSTTVVQRNYFDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKSEQ ID NO: 191 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPYAPLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK SEQ ID NO: 192EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMQWVRQMPGKGLEWMGAIYPGDGDIRYTQNFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARWEKSTTVVQRNYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKSEQ ID NO: 193 DVQMIQSPKSMSMSVGERVTLSCKASENVGTFVSWYQQKPDQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQSYSYPTFGSGTKLEMKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGECSEQ ID NO: 194 EIVLTQSPATLSLSPGERATLSCRASQSVNDYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQGGHAPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO: 195 DIQMTQSPSSLSASVGDRVTITCKASENVGTFVSWYQQKPGKAPKLLIYGASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQSYSYPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGECSEQ ID NO: 241 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFDTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGSLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKSEQ ID NO: 242 EVQLVESGGGLVQPGGSLRLSCAASGFAFSRYDMSWVRQAPGKGLESVAYISGGGANTYYLDNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLSYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK SEQ ID NO: 243QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFDTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGSLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGKSEQ ID NO: 244 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMQWVRQMPGKGLEWMGAIYPGDGDIRYTQNFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARWEKSTTVVQRNYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKSEQ ID NO: 245 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPYAPLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK SEQ ID NO: 246EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPYAPLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK SEQ ID NO: 247EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMQWVRQMPGKGLEWMGAIYPGDGDIRYTQNFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARWEKSTTVVQRNYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLGKSEQ ID NO: 248 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPYAPLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK

TABLE 44 Arm 1 VH/VL Arm 2 VH/VL Control with F405L with K409R mAbsubstitution substitution Isotype TM3B342 gp120 TM3B195 IgG2sigmaTM3B343 gp120 TM3B299 IgG2sigma B23B74 gp120 B23B32 IgG2sigma PTBB23gp120 TM3B291 IgG2sigma PD1B355 PD1B246 gp120 IgG2sigma PD1B356 PD1B248gp120 IgG2sigma

Example 16. Characterization of Bispecific PD-1/TIM-3 Antibodies

The generated antagonistic bispecific antibodies were tested in the CMVassay for their ability to enhance antigen-specific T cell responses.Functionality was measured by assessing CD137 expression on both CD4⁺and CD⁺ T cells and by IFN-γ and TNF-α levels in the culturesupernatants as described in Example 14. Table 45 and Table 46 summarizethe activity of bispecific PD-1/TIM-3 antibodies in this assay for thedifferent readouts. As shown in this table, select bispecific moleculesled to significant increases in CD137 expression on CD4⁺ and CD8⁺ Tcells and in levels of secreted IFN-γ and TNF-α. Overall, the PD-1/TIM-3bispecifics with huIgG2sigma Fc had the most robust activity, followedby those molecules with huIgG2 and then huIgG4.

TABLE 45 Statistical Significance CD4⁺CD137⁺ CD8⁺CD137⁺ mAb Avg p Avg pIsotype name value St Dev value St Dev IgG2sigma PTBB14 0.1144 0.15910.0002 0.0001 IgG2sigma PTBB15 0.0467 0.0988 0.0001 0.0000 IgG2sigmaPTBB16 0.0017 0.0023 0.0001 0.0000 IgG2sigma PTBB17 0.4148 0.5051 0.00010.0001 IgG2sigma PTBB24 0.0031 0.0051 0.0001 0.0000 IgG2 PTBB27 0.00090.0011 0.0001 0.0000 IgG2 PTBB28 0.0003 0.0002 0.0001 0.0000 IgG4PTBB18* 0.0353 0.0071 IgG4 PTBB20 0.6025 0.1710 0.0004 0.0004 IgG4PTBB21 0.1071 0.1372 0.0059 0.0081 *one p value reported

TABLE 46 Statistical significance IFN-γ TNF-α mAb Avg p Avg p Isotypename value St Dev value St Dev IgG2sigma PTBB14 0.0001 0.0000 0.01120.0157 IgG2sigma PTBB15 0.0001 0.0000 0.0005 0.0008 IgG2sigma PTBB160.0001 0.0000 0.0012 0.0016 IgG2sigma PTBB17 0.0001 0.0000 0.0001 0.0000IgG2sigma PTBB24 0.0001 0.0001 0.0008 0.0008 IgG2 PTBB27 0.0026 0.00300.3406 0.4757 IgG2 PTBB28 0.0001 0.0000 0.1437 0.1229 IgG4 PTBB18 0.0001#DIV/0! 0.0008 #DIV/0! IgG4 PTBB20 0.0544 0.0768 0.1754 0.2140 IgG4PTBB21 0.0174 0.0245 0.2685 0.1103 *one p value reported

Example 17. Anti-PD1 Antibodies Upregulate TIM-3 Expression on Tumors

Effect of anti-PD-1 antibody treatment in expression of TIM-3 on tumorswere evaluated in CT26 or MC38 colon carcinoma mouse model.

Balb/c mice were implanted subcutaneously with 1×10⁶ CT26 coloncarcinoma tumors. Seven days after tumor cell implant, tumors weremeasured and mice were randomized by tumor size. Treatment with PBS or10 mg/kg anti-mouse PD-1 antibodies (clone RMP1-14, BioXCell) began onday 7 after tumor cell implant and continued biweekly for the remainderof the study. To analyze T cell expression of TIM-3, tumors wereharvested at day 22 and dissociated using GentleMACS (Miltenyi).Staining for flow cytometry was carried out with Live/Dead and markersfor CD3, CD4, CD8 and TIM-3. Flow cytometry was performed on a LSRFortessa (BD). Data was analyzed using the Flow Jo software.

Wild-type C57Bl/6 female mice were implanted subcutaneously with 5×10⁵MC-38 colon carcinoma cells suspended in PBS. Tumors were measured andmice were randomized by tumor size (50-100 mm³) Treatment with PBS or 10mg/kg anti-mouse PD-1 (clone RMP1-14, BioXCell) began afterrandomization and continued biweekly for the remainder of the study. Toprofile tumor infiltrating T cells, tumors were harvested anddissociated using GentleMACS (Miltenyi) 12, 15, 19, or 22 days afterimplant.

Staining for flow cytometry was carried out with Live/Dead and markersfor CD45, Thy1, CD3, CD4, CD8, TIM-3, CD137, OX40, GITR, TIGIT. Flowcytometry data was collected on a LSR Fortessa (BD). Data was analyzedusing the FlowJo software (v9.9.4) and visualized with GraphPad Prism.Statistics were generated by GraphPad Prism.

Analysis of TIM-3 expression on CD8+ T cells isolated from CT26 tumorsat day 22 revealed an increase of TIM-3 expression in the PD-1 treatedsamples, compared to PBS control. FIG. 1A shows the mean fluorescentintensity of TIM-3 expression in the two treatment groups.

TIM-3 expression was also increased in MC-38 tumors in the anti-PD-1 mAbtreated samples when compared to PBS control. FIG. 1B shows thegeometric mean fluorescent intensity of TIM-3 expression in the CD8⁺ TILpopulation. FIG. 1C shows the percentage (%) relative frequency ofTIM-3⁺ CD8⁺ cells of total CD8⁺ TILs.

These data show that TIM-3 is upregulated in response to anti-PD-1treatment, supporting the rational for targeting TIM-3 in PD-1 treatedsubjects.

CD137, OX40 and GITR expression was also analyzed on CD8+ T cellsinfiltrating MC38 tumors isolated from mice treated with anti-mouse PD-1antibodies. These results showed that both the frequency and level(gMFI) of TNF family costimulatory receptors CD137, OX40 and GITRexpression was increased following PD-1 blockade. FIG. 2A and FIG. 2Bshow the gMFI and relative frequency of CD137 expression on CD8 TILs,respectively. FIG. 3A and FIG. 3B show the gMFI and relative frequencyof OX40 expression on CD8 TILs, respectively, and FIG. 4A and FIG. 4Bshow the gMFI and relative expression of GITR on CD8 TILs, respectively.

These data support the rational for targeting CD137, OX40 and/or GITR inPD-1 treated subjects.

Example 18. Activity of Anti-TIM-3 Antibodies Following PD-1 Blockade

The activity of anti-TIM-3 antibodies was also tested followinganti-PD-1 antibody blockade in the CMV assay. In these experiments,PBMCs from one normal donor (CMV-sera positive) were incubated with pp65peptide pools and anti-PD-1 antibodies for 5 days. On day 5,supernatants were harvested and cells were re-stimulated with pp65peptide pool in the presence of either anti-TIM-3 or anti-PD-1 antibody.IFN-γ levels in the supernatant were measured 24 hours later. Treatmentwith anti-TIM-3 antibodies after 5 days of anti-PD-1 blockade resultedin a significant increase of IFN-γ levels. This effect was significant(p=0.0183) compared to continued anti-PD-1 treatment. In the experiment,anti-TIM-3 antibody TM3B403 and anti-PD-1 antibody PD1B244 were used.FIG. 5 shows the increased IFN-γ levels in the CMV assay, where PBMCswere treated with anti-TIM-3 antibody TM3B105 following 5 days oftreatment with anti-PD-1 PD1B244. Values represent average of sixbiological replicates used for each condition.

Example 19. Epitope Mapping of Anti-TIM-3 Antibodies

Solution hydrogen/deuterium exchange-mass spectrometry (HDX-MS) wasperformed to identify the binding epitopes of TMB403 and TMB291. For theexperiments, the VH and the VL of TM3B403 and TM3B291 were cloned asIgG1 Fabs with a hexahistidine tag in the C-terminus. The Fabs, weregenerated from transient transfections of HEK293 Expi cells insuspension shake flasks. TIM-3 IgG1 Fc Chimera, Ser22-Arg200 (Accession#Q8TDQ0), produced in Mouse myeloma cell line (NS0 derived) from R&DSystems (Catalog #2365-TM) was used.

For H/D exchange, the procedures used to analyze the Fab perturbationwere similar to those described previously (Hamuro et al., BiomolecularTechniques 14: 171-182, 2003; Horn et al., Biochemistry 45: 8488-8498,2006) with some modifications. Briefly, deglycosylated human TIM-3/Fcfusion protein or deglycosylated human TIM-3-Fc plus Fab mixture wasincubated with deuterium oxide labeling buffer at 0° C. for varioustimes up to 2 hours. Deuterium exchange was quenched by adding guanidinehydrochloride and the quenched sample was subjected to on-column pepsindigestion and LC-MS analysis. The mass spectra were recorded in MS onlymode. For the calculation of deuterium incorporation, the mass spectrafor a given peptide were combined across the extracted ion chromatogrampeak and the weighted average m/z was calculated. The mass increase fromthe mass of the native peptide (0 min) to the weighted averaged masscorresponds to the level of deuterium incorporation. About 98.4% of theprotein could be mapped to specific peptides.

The deuterium levels at the identified peptides were monitored from themass shift on LC-MS. The selected deuterium buildup curves, which showsignificant difference in deuterium levels and/or slopes, over exchangetime for the peptides were plotted. Deglycosylated human Tim-3/Fc fusionprotein showed significant reduction in deuterium uptakes upon bindingto TM3B403 at sequences ₃₂WGKGACPVFECGNVVL₄₇, (SEQ ID NO: 261) and uponbinding to TM3B291 at sequences ₉₀RIQIPGIMNDEKF₁₀₂. (SEQ ID NO: 262).These regions with significant reduction in deuterium uptakes uponbinding to Fabs can thus be regarded as main epitopes of the mAbs.

A segment, ₅₀DERDVNY₅₆, (SEQ ID NO: 263) demonstrated modest reductionin deuterium exchange upon binding to TM3B403 or TM3B291. This regionmay be also considered as a potential epitope for both antibodies.

The major binding epitopes for TM3B403 or TM3B291 are different.However, they may share the similar modest protection region,₅₀DERDVNY₅₆, (SEQ ID NO: 263) based on the HDX mapping results. To helpassess if this region contributes to common binding epitope region forboth Fab molecules, competition ELISA was performed. Recombinant humanTim-3/Fc protein was directly coated on plates which were then blockedand washed. A mixture of Ruthenium (Ru)-labeled TM3B291 Fab which waspre-incubated with different concentrations of unlabeled TM3B105 orTM3B291. Plates were incubated, washed and MSD Read Buffer T wasdispensed into each well followed by reading with a SECTOR Imager 6000(Meso Scale Discovery, Gaithersburg, Md.).

The competition analysis demonstrated that that TM3B403 competed forbinding to TIM-3 with TM3B291. This result could indicate that themodestly protected region, DERDVNY (SEQ ID NO: 263) is part of theepitope for both antibodies or that the antibodies may be stericallyblocking each other's binding due to the close proximity of theirepitopes.

Example 20. TIM-3 Blockade Increases TIGIT Expression on CD8⁺ TILs

Effect of anti-TIM-3 antibody treatment on expression of TIGIT in tumorswas evaluated in CT26 and MC38 colon carcinoma mouse models. The studieswere conducted as described in Example 17 except that 10 mg/mlanti-TIM-3 antibody RMT3-23 was used.

TIGIT expression on CD8+ TILs (FIG. 19A, FIG. 20A) and relativefrequency of TIGIT+ TILs f (FIG. 19B, FIG. 20B) were elevated in bothCT26 (FIG. 19A, FIG. 19B) and MC38 (FIG. 20A, FIG. 20B) tumor modelsfollowing TIM-3 blockage.

Example 21. TIM-3 Expression is Increased after Ex Vivo PD-1 Blockade inMelanoma Patient PBMC

PBMCs from treatment naïve melanoma patients were stimulated withmelanoma antigen peptide pools (NY-ESO, gp100, MART-1) in the presenceof anti-PD-1 or anti-TIM-3 function blocking antibodies. Expression ofTIM-3 was evaluated on peptide-restimulated cells on day 6. Resultsshowed significant increases in the frequency of TIM-3+ CD8+ T cells inthe anti-PD-1 treated samples compared to controls or TIM-3 treatedPBMCs (FIG. 21).

On day 0, frozen PBMCs from treatment naïve melanoma patients wererapidly thawed in a 37° C. water bath. Cells were thawed, washed andcounted in complete RPMI media (RPMI+10% FBS+1% sodium pyruvate+1%NEAA+1% pen/strep). Cells were plates at 200,000 cells per well in a 96well, U-bottom plate in the presence or absence of anti-PD-1 oranti-TIM-3 function blocking antibodies (PD1B244 and TM3B403,respectively) and 1)(g/mL of melanoma antigen peptide pools (NY-ESO,gp100, MART-1) for 6 days at 37 C. Cells were restimulated with thepeptide pool at day 6 and analyzed by flow cytometry for expression ofPD-1 and TIM-3 as well as T cell activation and proliferation markers.

Example 22. Anti-TIM-3 Antibodies Increase the Frequency of Activated NKCells in IL-2 Stimulated PBMCs

The effects of anti-TIM-3 antibody TM3B403 on the frequency of activatedNK cells was determined in assays where human PBMCs were stimulated withIL-2 (20 U). Frequency of CD69 and CD25, markers of NK cell activation,were evaluated by flow cytometry 48 hours post-treatment at a range ofmAb concentrations. TM3B403 increased the frequency of activated NKcells when the activation was assessed by percentage of CD69 positivecells (FIG. 22A) or percentage of CD25 positive cells (FIG. 22B).

What is claimed:
 1. An isolated antagonistic antibody or anantigen-binding portion thereof specifically binding PD-1, comprising aheavy chain complementarity determining region (HCDR) 1 a HCDR2 and aHCDR3 of SEQ ID NOs: 10, 14 and 17, respectively, and a light chaincomplementarity determining region (LCDR)-1, a LCDR2 and a LCDR3 of SEQID NOs: 23, 26 and 32, respectively.
 2. The antibody or theantigen-binding portion thereof of claim 1, comprising a heavy chainvariable region (VH) of SEQ ID NO: 48 and a light chain variable region(VL) of SEQ ID NO:
 56. 3. The antibody or the antigen-binding portionthereof of claim 1, wherein the antibody is human or humanized.
 4. Theantibody or the antigen-binding portion thereof of claim 3, wherein theantibody is a) an IgG1 isotype, optionally comprising one, two, three,four, five, six, seven, eight, nine or ten substitutions in an Fcregion; b) an IgG2 isotype, optionally comprising one, two, three, four,five, six, seven, eight, nine or ten substitutions in the Fc region; c)an IgG3 isotype, optionally comprising one, two, three, four, five, six,seven, eight, nine or ten substitutions in the Fc region; d) an IgG4isotype, optionally comprising one, two, three, four, five, six, seven,eight, nine or ten substitutions in the Fc region; e) an IgG1 isotypecomprising L234A, L235A, G237A, P238S, H268A, A330S and P331 Ssubstitutions; f) an IgG2 isotype comprising V234A, G237A, P238S, H268A,V309L, A330S and P331 S substitutions; g) an IgG4 isotype comprisingF234A, L235A, G237A, P238S and Q268A substitutions; h) an IgG1 isotypecomprising L234A, L235A or L234A and L235A substitutions; i) an IgG4isotype comprising F234A, L235A or F234A and L235A substitutions; j) anIgG2 isotype comprising a V234A substitution; k) an IgG4 isotypecomprising a S228P substitution; or l) an IgG4 isotype comprising S228P,F234A and L235A substitutions, wherein residue numbering is according tothe EU Index.
 5. The antibody or the antigen-binding portion thereof ofclaim 2, comprising a heavy chain (HC) of SEQ ID NO: 72 and a lightchain (LC) of SEQ ID NO:
 73. 6. The antibody or the antigen-bindingportion thereof of claim 2, wherein the antibody is a bispecificantibody, optionally binding PD-Ll (SEQ ID NO: 5), PD-L2 (SEQ ID NO: 8),LAG-3 (SEQ ID NO: 293), TIM-3 (SEQ ID NO: 138), CEACAM-1 (SEQ ID NO:296), CEACAM-5 (SEQ ID NO: 307), OX-40 (SEQ ID NO: 279), GITR (SEQ IDNO: 271), CD27 (SEQ ID NO: 280), VISTA (SEQ ID NO: 286), CD137 (SEQ IDNO: 281), TIGIT (SEQ ID NO: 301) or CTLA-4 (SEQ ID NO: 292).
 7. Apharmaceutical composition comprising the antibody or theantigen-binding portion thereof of claim 2 and a pharmaceuticallyaccepted carrier.
 8. An isolated antagonistic antibody specificallybinding PD-1 or an antigen-binding portion thereof, comprising a) theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQID NOs: 66, 67, 68, 69, 70 and 71, respectively; b) the VH of SEQ ID NO:64 and the VL of SEQ ID NO: 65; and/or c) the HC of SEQ ID NO: 74 andthe LC of SEQ ID NO:
 75. 9. A pharmaceutical composition comprising theantibody or the antigen-binding portion thereof of claim 8 and apharmaceutically accepted carrier.
 10. A kit comprising the antibody ofclaim
 2. 11. The antibody or the antigen-binding portion thereof ofclaim 6, wherein the bispecific antibody comprises a first domainspecifically binding PD-1 and a second domain specifically bindingTIM-3.
 12. The antibody or the antigen-binding portion thereof of claim11, wherein a) the first domain comprises the HCDR1, the HCDR2, theHCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 10, 14, 17, 23,26 and 32, respectively or SEQ ID NOs: 66, 67, 68, 69, 70 and 71,respectively; and b) the second domain comprises the HCDR1, the HCDR2,the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 97, 105,115, 124, 133 and 143, respectively; or SEQ ID NOs: 91, 99, 108, 118,127 and 136, respectively.
 13. The antibody or the antigen-bindingportion thereof of claim 12, wherein the bispecific antibody binds TIM-3within TIM-3 residues 32-47 (WGKGACPVFECGNVVL) (SEQ ID NO: 261),optionally further binding within TIM-3 residues 50-56 (DERDVNY) (SEQ IDNO: 262).
 14. The antibody or the antigen-binding portion thereof ofclaim 13, wherein a. the first domain comprises the HCDR1, the HCDR2,the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 10, 14, 17,23, 26 and 32, respectively, and the second domain comprises the HCDR1,the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:91, 99, 108, 118, 127 and 136, respectively; b. the first domaincomprises the VH of SEQ ID NO: 48 and the VL of SEQ ID NO: 56, and thesecond domain comprises the VH of SEQ ID NO: 146 and the VL of SEQ IDNO: 156; c. the antibody comprises a first heavy chain (HCl), a firstlight chain (LC1), a second heavy chain (HC2) and a second light chain(LC2) of SEQ ID NOs: 186, 188, 191 and 194, respectively; d. theantibody comprises the HCl, the LC1, the HC2 and the LC2 of SEQ ID NOs:186, 188, 248 and 194, respectively; e. the antibody comprises the HCl,the LC1, the HC2 and the LC2 of SEQ ID NOs: 241, 188, 245 and 194,respectively; and/or f. the antibody comprises the HCl, the LC1, the HC2and the LC2 of SEQ ID NOs: 243, 188, 246 and 194, respectively.
 15. Theantibody or the antigen-binding portion thereof of claim 12, wherein thebispecific antibody binds TIM-3 within TIM-3 residues 90-102(RIQIPGIMNDEKF) (SEQ ID NO: 263), optionally further binding withinTIM-3 residues 50-56 (DERDVNY) (SEQ ID NO: 262).
 16. The antibody or theantigen-binding portion thereof of claim 15, wherein a. the first domaincomprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and theLCDR3 of SEQ ID NOs: 10, 14, 17, 23, 26 and 32, respectively, and thesecond domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 of SEQ ID NOs: 97, 105, 115, 124, 133 and 143,respectively; b. the first domain comprises the VH of SEQ ID NO: 48 andthe VL of SEQ ID NO: 56, and the second domain comprises the VH of SEQID NO: 172 and the VL of SEQ ID NO: 173; c. the antibody comprises aHCl, a LC1, a HC2 and a LC2 of SEQ ID NOs: 186, 188, 192 and 195,respectively; d. the antibody comprises the HCl, the LC1, the HC2 andthe LC2 of SEQ ID NOs: 241, 188, 244 and 195, respectively; and/or e.the antibody comprises the HCl, the LC1, the HC2 and the LC2 of SEQ IDNOs: 243, 188, 247 and 195, respectively.
 17. The antibody or theantigen-binding portion thereof of claim 11, wherein the bispecificantibody is a. an IgG1 isotype, optionally comprising one, two, three,four, five, six, seven, eight, nine or ten substitutions in an Fcregion; b. an IgG2 isotype, optionally comprising one, two, three, four,five, six, seven, eight, nine or ten substitutions in an Fc region; c.an IgG3 isotype, optionally comprising one, two, three, four, five, six,seven, eight, nine or ten substitutions in an Fc region; d. an IgG4isotype, optionally comprising one, two, three, four, five, six, seven,eight, nine or ten substitutions in an Fc region; e. an IgG1 isotypecomprising L234A, L235A, G237A, P238S, H268A, A330S and P331 Ssubstitutions; f. an IgG2 isotype comprising V234A, G237A, P238S, H268A,V309L, A330S and P331 S substitutions; g. an IgG4 isotype comprisingF234A, L235A, G237A, P238S and Q268A substitutions; h. an IgG1 isotypecomprising L234A, L235A or L234A and L235A substitutions; i. an IgG4isotype comprising F234A, L235A or F234A and L235A substitutions; j. anIgG2 isotype comprising a V234A substitution; k. an IgG4 isotypecomprising a S228P substitution; l. an IgG4 isotype comprising S228P,F234A and L235A substitutions; m. an IgG2 isotype comprising F405L andR409K substitutions; or n. an IgG1 isotype comprising F405L and K409Rsubstitutions, wherein residue numbering is according to the EU Index.18. A pharmaceutical composition comprising the bispecific antibody ofclaim 12 and a pharmaceutically accepted carrier.
 19. An isolatedantagonistic antibody specifically binding PD-1 or an antigen-bindingportion thereof, comprising a HCDR1, a HCDR2 and a HCDR3 of SEQ ID NOs:11, 15 and 18, respectively and a LCDR1, a LCDR2 and a LCDR3 of SEQ IDNOs: 20, 30 and 32, respectively.
 20. The antibody or theantigen-binding portion thereof of claim 19, comprising a VH and a VL ofSEQ ID NOs: 45 and 60, respectively.
 21. The antibody or theantigen-binding portion thereof of claim 20, wherein the antibody is anIgG4 isotype comprising a S228P substitution.